Myalgic Encephalomyelitis/Chronic Fatigue Syndrome
Phase: NA
Conditions: Chronic Fatigue Syndrome
Sponsor: National Institute of Neurological Disorders and Stroke (NINDS)

The attached includes: 
Protocol: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome 
•
Statistical Plan included within the protocol
NCT02669212 
Document Date:  11/10/2020 

M E/ C F S at NI H
M yal gic E nce p hal o m yelitis/ C hr o nic Fati g ue S y n dr o me 
M E/ C F S at NI H
Pr ot oc ol N u m ber: 1 6- N- 0 0 5 8
Date of T his S u b missi o n/ Versi o n: 1 1/ 1 0/ 2 0 2 0, Versi o n 1 8. 0 
Pri nci pal I n vesti gat or
H u ma n Researc h Pr otecti o ns Pr o gra m I n vesti gat or a n d Staff Trai ni n g:
F or t his pr ot oc ol, t he f oll o wi n g “J ust i n ti me” h u ma n s u bjects pr otecti o n trai ni n g c o urses 
are re q uire d f or i n vesti gat ors a n d staff: N o ne
T otal re q ueste d accr ual: 
3 4 6
( 5 0) P ost-i nfecti o us M yal gic E nce p hal o m yelitis/ C hr o nic Fati g ue S y n dr o me partici pa nts
( 5 0) Healt h y V ol u nteers
( 5 0)  Rec o vere d C O VI D- 1 9 i nfecti o n —as y m pt o matic partici pa nts
( 1 7 6) F oc us Gr o u p partici pa nts
( 2 0) Tec h nical De vel o p me nt s u b-st u d y
Pr oject Uses I o nizi n g Ra diati o n:   N o
Yes
Me dicall y-i n dicate d  o nl y
 Researc h-relate d o nl y
B ot h
C o vere d Pr ot oc ol Re q uiri n g D E C Cleara nce ( per S O P 2 1)  N o
 Yes 
A p pr o ve d f or S h ort F or m C o nse nt Pr ocess f or N o n- E n glis h S pea kers  N o Yes 
Flesc h- Ki ncai d rea di n g le vel of M E/ C F S, H V c o nse nt f or m: 8. 9
Flesc h- Ki ncai d rea di n g le vel of C O VI D- 1 9 H V c o nse nt f or m: 9. 2
Flesc h- Ki ncai d rea di n g le vel of F oc us Gr o u p A c o nse nt f or m: 8. 6
C N S I R B Pr ot oc ol Te m plate ( 3. 1 1. 1 5)
Na me, De gree
Bra nc h/I nstit ute
Bl d g/ R m
P h o ne
E- mail
A vi n dra Nat h, 
M D
SI N S/ NI N D S
1 0/ 7 C-
1 0 3
3 0 1- 4 9 6- 1 5 6 1
nat ha @ ni n ds. ni h. g o v
I N D/I D E
 N o
Yes
D ura ble P o wer of Att or ne y
 N o
□ Y es
M ulti-i nstit uti o nal Pr oject
 N o
□ Y es
Data a n d Safet y M o nit ori n g B oar d
 N o
□ Y es
Tec h n ol o g y Tra nsfer A gree me nt
 N o
□ Y es
Sa m ples are bei n g st ore d
□ N o
 Yes

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Flesch-Kincaid reading level of Focus Group B consent form: 9.0
Flesch-Kincaid reading level of Technical Development Substudy consent form: 9.7 
Flesch-Kincaid reading level of Genetic consent form: 10.5

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Précis:
Objective The primary objective is to explore the clinical and biological phenotypes of 
post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS). The 
secondary objective is to explore the pathophysiology of fatigue and post-exertional 
malaise (PEM).
Study population Up to 346 persons will be enrolled as part of this protocol. Up to 150 
persons aged 18-60 will be part of 3 study groups: 50 ME/CFS patients whose fatigue 
began after an infection, 50 non-fatigued participants with a documented history of a full 
recovery from a COVID-19 infection, and 50 healthy volunteers. The study has a target 
of completing all study procedures on 20 enrolled participants in each group. Up to an 
additional 176 persons reporting a community diagnosis of ME/CFS will be enrolled into 
focus groups to discuss the experience of post-exertional malaise. Up to an additional 10 
healthy volunteers and 10 ME/CFS patients may be enrolled to refine the protocol’s 
electrophysiological and neuroimaging techniques.
Design This is a single-center, exploratory, cross-sectional study of PI-ME/CFS. 
Participants will have a phenotyping visit, which will encompass a 2-5 day long inpatient 
admission at the NIH Clinical Center. Case status for ME/CFS participants will be 
determined after the phenotyping visit by a case adjudication process utilizing an expert 
physician committee and published guidelines. Adjudicated participants meeting 
inclusion criteria will be invited back to participate in an exercise stress visit, which will 
encompass a 5-10 day long inpatient admission. Detailed subjective and objective 
measurements and biological specimens will be serially collected before and up to 96 
hours after a peak exercise test capable of inducing post-exertional malaise during this 
visit. All procedures will be completed on all three study groups to allow for optimal 
inter-group comparisons.
Outcome measures
The primary purpose of this protocol is to perform exploratory analysis of collected 
samples for the generation of new hypotheses regarding ME/CFS. The types of analyses 
to be performed will be wide ranging. Planned areas of focus include:
1. Characterization of the immune system and inflammatory signaling in collected 
samples at baseline and following maximal exercise exertion.
2. Characterization of the pattern of microbiome in collected samples at baseline and 
following maximal exercise exertion.
3. Characterization of bioenergetics, autonomic, and metabolic function in collected 
samples at baseline and following maximal exercise exertion.
4. Characterization of physical and cognitive fatigue using functional magnetic 
resonance imaging and transcranial magnetic stimulation at baseline and 
following maximal exercise exertion.
5. Characterization of neurocognition at baseline and following maximal exercise 
exertion.

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6. Characterization of brain function and connectivity at baseline and following 
maximal exercise exertion.
7. Characterization of autonomic function at baseline and following maximal 
exercise exertion.
8. Characterization of gene expression profiles in collected samples at baseline and 
following maximal exercise exertion.

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Contents
Précis: ..................................................................................................................................2
List of Abbreviations ...........................................................................................................8
1
Introduction ................................................................................................................10
1.1
HISTORY...................................................................................................................................................10
1.2
DEFINITION............................................................................................................................................11
1.3
ETIOLOGY................................................................................................................................................11
1.4
EPIDEMIOLOGY ....................................................................................................................................13
1.5
DIAGNOSTIC CHALLENGES.............................................................................................................13
1.6
SOCIO-ECONOMIC BURDEN............................................................................................................14
1.7
DISABILITY.............................................................................................................................................14
1.8
MORTALITY............................................................................................................................................14
2
Study Objectives.........................................................................................................15
2.1
Primary objective.................................................................................................................................15
2.2
Secondary objective............................................................................................................................15
3
Subjects ......................................................................................................................15
3.1
Description of study populations..................................................................................................15
3.2
Inclusion criteria ..................................................................................................................................16
3.2.1
Inclusion criteria for all participants .................................................................................16
3.2.2
Additional inclusion criteria for participants with PI-ME/CFS for the 
phenotyping visit ...........................................................................................................................................16
3.2.3
Additional inclusion criteria for participants with PI-ME/CFS for the exercise 
stress visit .........................................................................................................................................................17
3.2.4
Additional inclusion criteria for healthy volunteer group........................................17
3.2.5
Additional inclusion criteria for Documented Lyme Infection – Asymptomatic 
group
17
3.3
Exclusion criteria .................................................................................................................................18
3.3.1
Exclusion criteria for all participants ................................................................................18
3.3.2
Additional exclusion criteria for participants with PI-ME/CFS for phenotyping 
visit
19
3.3.3
Additional exclusion criteria for participants with PI-ME/CFS for exercise 
stress visit .........................................................................................................................................................19
3.3.4
Additional exclusion criteria for healthy volunteer group.......................................19
3.3.5
Additional exclusion criteria for Documented Lyme Infection – Asymptomatic 

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group
19
4
Study Design and Methods.........................................................................................19
4.1
Study overview......................................................................................................................................19
4.2
Recruitment............................................................................................................................................21
4.3
Focus Groups..........................................................................................................................................22
4.31
Focus Group Study A...........................................................................................................................22
4.31.1
Inclusion criteria for focus group A participants..........................................................22
4.31.2
Exclusion criteria for focus group participants.............................................................23
4.32
Focus Group Study B...........................................................................................................................24
4.4
Technical Development Sub-Study...............................................................................................27
4.5
Phenotyping Visit (Visit 1: Phenotyping) ..................................................................................29
4.6 Study procedures: Exercise Stress visit (Visit 2)...........................................................................43
4.6.1 : Updated History and Physical Examination ..........................................................................44
4.6.2 : Intravenous line placement ..........................................................................................................44
4.6.3 : Blood collection..................................................................................................................................44
4.6.4 : Urine collection ..................................................................................................................................44
4.6.5 : Saliva sample collection..................................................................................................................44
4.6.6 : Patient-reported outcome.............................................................................................................45
4.6.7 : Physical activity monitoring.........................................................................................................47
4.6.8 : Metabolic Chamber...........................................................................................................................47
4.6.9 : Body Composition Measurement by Dual Energy X-ray Absorptiometry................48
4.6.10 : Metabolic Diet and Nutritional Assessment........................................................................48
4.6.11 Resting Energy Expenditure Measurements by Ventilated Hood...............................48
4.6.12 Peak Exercise Testing.....................................................................................................................49
4.6.13 : Qualitative measurement of Post-Exertional Malaise.....................................................49
4.6.14 : Blood measurements before and after CPET......................................................................50
4.6.15 : Neurocognitive testing.................................................................................................................50
4.6.16 : Holter monitoring...........................................................................................................................50
4.6.17 : fMRI ......................................................................................................................................................50
4.6.18 : Effort-Expenditure for Rewards Task (EEfRT)..................................................................52
4.6.19 : Lumbar puncture............................................................................................................................53
4.6.20 : Stool Sample Collection................................................................................................................53
4.6.21 : Sleep Electrocephalogram..........................................................................................................53

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5
Descriptions of Optional Produres .............................................................................53
5.1.1
Sedation during LP.....................................................................................................................53
5.1.2
Fluoroscopic-guided LP...........................................................................................................54
5.1.3
Medical, Psychiatric, and Rehabilitation Consultation...............................................54
5.1.4
Whole genome sequencing ....................................................................................................54
5.1.5
Skin Biopsies ................................................................................................................................54
5.1.6
Muscle Biopsy ..............................................................................................................................54
5.1.7
Functional Magnetic Resonance Imaging (fMRI)..........................................................55
5.1.8
Transcranial Magnetic Stimulation (TMS) ......................................................................55
5.1.9
Metabolic Chamber wearing EEG cap and accelerometers......................................56
6
End of participation....................................................................................................56
7
Management of Data and Samples.............................................................................57
7.1
Storage......................................................................................................................................................57
7.2
Data (including genomic data) and sample sharing plan ...................................................57
8
Additional Considerations..........................................................................................58
8.1
Research with investigational drugs or devices.....................................................................58
8.2
Gene therapy..........................................................................................................................................59
9
Risks and Discomforts................................................................................................59
9.1
Risks of history and physical examination................................................................................59
9.2
Risks of IV insertion............................................................................................................................59
9.3
Risks of blood collection ...................................................................................................................59
9.4
Risks of HIV testing .............................................................................................................................59
9.5
Risks of urine collection....................................................................................................................59
9.6
Risks of symptom assessment/patient reported outcome measures...........................60
9.7
Risks of psychological assessments and psychological inventories ..............................60
9.8
Risks of neurocognitive testing......................................................................................................60
9.9
Risks of Occupational therapy evaluation.................................................................................61
9.10
Risks of MRI............................................................................................................................................61
9.11
Risks of muscle strength testing....................................................................................................62
9.12
Risks of activity monitoring and fatigue diary ........................................................................63
9.13
Risks of nutritional assessment and metabolic diet .............................................................63
9.14
Risks of saliva collection ...................................................................................................................63
9.15
Risks of buccal swab collection......................................................................................................63

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9.16
Risks of stool sample collection.....................................................................................................63
9.17
Risks of Holter monitoring...............................................................................................................63
9.18
Risks of LP...............................................................................................................................................63
9.19
Risks of sedation for LP.....................................................................................................................63
9.20
Risks of fluoroscopic-guided LP.....................................................................................................63
9.21
Risks of autonomic testing...............................................................................................................64
9.22
Risks of cytapheresis..........................................................................................................................64
9.23
Risks of immune cell blood draw..................................................................................................64
9.24
Risks of medication washout ..........................................................................................................64
9.25
Risks of metabolic chamber.............................................................................................................64
9.26
Risks of DEXA.........................................................................................................................................64
9.27
Risks of resting energy expenditure measurements by ventilated hood ....................65
9.28
Risks of peak exercise testing.........................................................................................................65
9.29
Risks of Qualitative Interviews ......................................................................................................65
9.30
Blood measurements before and after CPET...........................................................................65
9.31
Risks of Effort-Expenditure for Rewards Task........................................................................65
9.32
Risks of TMS ...........................................................................................................................................65
9.33
Risks of sleep electroencephalogram..........................................................................................66
9.34
Risks of Medical, Psychiatric, Sleep Service and Rehabilitation Consultation...........66
9.35
Risks of genetic testing......................................................................................................................66
9.36
Risks of Skin Biopsies.........................................................................................................................67
9.37
Risks of Muscle Biopsy.......................................................................................................................67
10
Subject Safety Monitoring......................................................................................67
11
Outcome Measures .................................................................................................67
12
Statistical Analysis..................................................................................................67
12.1
Analysis of data/ study outcomes.................................................................................................67
12.2
Power analysis ......................................................................................................................................69
13
Human Subjects Protection.....................................................................................69
13.1
Subject selection...................................................................................................................................69
13.2
Justification for exclusion of children..........................................................................................70
13.3
Justification for exclusion of pregnant women .......................................................................70
13.4
Justification for exclusion of women who have given birth in the past year..............70
13.5
Justification for exclusion of those who lack consent capacity ........................................70

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13.6
Justification of exclusion of non-English speakers ................................................................70
13.7
Justification for inclusion of employees.....................................................................................70
13.8
Justification for sensitive procedures .........................................................................................71
13.9
Safeguards for vulnerable populations and sensitive procedures .................................71
14
Anticipated Benefit:  Indirect .................................................................................72
15
Consent Documents and Process............................................................................72
15.1
Consent procedures............................................................................................................................72
15.2
Consent documents.............................................................................................................................73
16
Data and Safety Monitoring....................................................................................73
16.1
Data and safety monitor....................................................................................................................73
16.2
Data and safety monitoring plan...................................................................................................73
16.3
Criteria for stopping the study or suspending enrollment or procedures..................73
17
Quality Assurance...................................................................................................73
17.1
Quality assurance monitor...............................................................................................................73
17.2
Quality assurance plan.......................................................................................................................73
18
Reporting of Unanticipated Problems, Adverse Events and Protocol Deviations 74
19
Alternatives to Participation ...................................................................................74
20
Privacy ....................................................................................................................74
21
Confidentiality ........................................................................................................74
21.1
For research data and investigator medical records............................................................74
21.2
For stored samples..............................................................................................................................74
21.3
Special precautions .............................................................................................................................74
22
Conflict of Interest..................................................................................................74
22.1
Distribution of NIH guidelines........................................................................................................74
22.2
Conflict of interest ...............................................................................................................................75
23
Technology Transfer...............................................................................................75
24
Research and Travel Compensation .......................................................................75
25
Attachments/ Appendices .......................................................................................78
25.1
Schedule of Events...............................................................................................................................79
25.2
Questionnaires ......................................................................................................................................82
25.3
Case Report Forms (CRFs)...............................................................................................................82
25.4
Focus Group Forms .............................................................................................................................82
25.5
Advertisements.....................................................................................................................................82

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25.6
Website Language................................................................................................................................82
25.7
Screening Questionnaires.................................................................................................................82
25.8
Thank you letters .................................................................................................................................82
25.9
Letter for Retroactive Increase in Compensation for Technical Development 
Substudy.................................................................................................................................................................82
25.10
Adjudication Board Members....................................................................................................82
26
Consent Forms........................................................................................................82
27
References...............................................................................................................83
List of Abbreviations
AE
adverse event
AI
associate investigator
ANA
anti-nuclear antibody
AT
anaerobic threshold
BPI
brief pain inventory
CBC
complete blood count
CC
cubic centimeter
CDC
Centers for Disease Control and Prevention 
CNS
central nervous system
CFS
chronic fatigue syndrome
CO
carbon monoxide
CO2
carbon dioxide 
CO2HB
carbaminohemoglobin
COPD
chronic obstructive pulmonary disease 
CPET
cardiopulmonary exercise test
CSF
cerebrospinal fluid
CRP
c-reactive protein
CTCAE
common terminology criteria for adverse events 
DEXA
dual energy X-ray absorptiometry
DSM
diagnostic and statistical manual of mental disorders 
DoH
Declaration of Helsinki
EBV
Epstein-Barr Virus
EEfRT
Effort-Expenditure for Rewards Task 
ELISA
enzyme-linked immunosorbent assay 
ESR
erythrocyte sedimentation rate
EKG
electrocardiogram
FACIT
functional assessment of chronic illness therapy 
FDA
Food and Drug Administration
fMRI
functional magnetic resonance imaging 
FSQ
fibromyalgia survey questionnaire
FWA
federal-wide assurance

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HV
healthy volunteer
HHV-6
human herpesvirus-6
HIV
human immunodeficiency virus 
HSPU
human subjects protection unit 
ID
identification
INR
international normalized ratio
IRB
Institutional Review Board
ISI
inter-stimulus interval
LAI
lead associate investigator
LIP
Licensed Independent Practitioner
LP
lumbar puncture
MASQ
multiple ability self-report questionnaire 
MD
medical doctor
ME
myalgic encephalomyelitis
MFI
multidimensional fatigue inventory
ML
milliliter
MPQ
McGill pain questionnaire 
mREM
milliRoentgen Equivalent in Man 
MRI
magnetic resonance imaging
MSEC
millisecond
NIH
National Institutes of Health
NIH-BFI
National Institutes of Health_ brief fatigue inventory 
NIMH
National Institute of Mental Health
NINDS
National Institute of Neurological Disorders and Stroke 
NINR
National Institute of Nursing Research
NMR
nuclear magnetic resonance
NPS
neuropathic pain scale
O2
oxygen
O2HB
oxyhemoglobin
OHSRP
Office of Human Subjects Research Protection 
PBMC
peripheral blood mononuclear cell
PCR
polymerase chain reaction
PEM
post exertional malaise
PI
principal investigator
PI-ME/CFS
Post-Infectious Myalgia Encephalomyelitis/Chronic Fatigue Syndrome 
PPI
pixels per inch
PROMIS
patient reported outcomes measurement information system 
PROMIS-F
patient reported outcomes measurement information system-fatigue 
PRPL
patient recruitment and public liaison
PSQI
Pittsburgh sleep quality index
PT
prothrombin time
PTT
partial thromboplastin time
QA
quality assurance
RF
rheumatoid factor
RPR
rapid plasma reagin
SAE
serious adverse event

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SCID
structural clinical interview and diagnosis 
SEID
systemic exertion intolerance disease
SF-36
short-form 36
SOP
standard operating procedure
SSS
symptom severity score
T
tesla
TMS
transcranial magnetic stimulation
TSH
thyroid stimulating hormone
T3
triiodothyronine
T4
free thyroxine
V02
oxygen consumption
WPI
widespread pain index
1
Introduction
1.1 HISTORY
Chronic fatigue syndrome (CFS), myalgic encephalomyelitis (ME), and systemic 
exertion intolerance disease (SEID) are names used to describe a disorder characterized 
by persistent and disabling fatigue, exercise intolerance, dyscognition, and physical 
discomfort often following an acute infection. These disorders were described as part of 
epidemics, with benign ME being defined after an epidemic at the Royal Free hospital in 
London, England (Royal Free Syndrome)1,2 and CFS being defined after an epidemic in 
Incline Village, Nevada.3 These diagnoses are historically controversial, with scientists 
and physicians disagreeing over whether these disorders are better considered to be a 
distinct disease entity with a clear organic basis4 or related to psychological factors and 
social contagion.5,6 Although various infectious agents have been implicated, including 
brucella7, poliovirus8, Epstein-Barr Virus (EBV)9, Human T-cell Lymphoma Virus II 
(HTLV-II)10, and the Xenotropic Murine Leukemia Virus-Related Virus (XMRV)11, no 
infection has yet been found to be necessary to cause ME/CFS. This is in contrast to other 
types of known infectious encephalitis, as has been demonstrated with strains of 
herpesvirus, enterovirus, alphavirus, flavivirus, buynavirus and even evidenced by the 
discovery of a submicroscopic, filter-passing agent during the encephalitis lethargica 
epidemic of 1916-1927.12,13 However, epidemiological evidence that viral infections are 
sufficient to cause ME/CFS is fairly strong, with prospective studies estimating the 
incidence of ME/CFS after several different documented infections to be between 10- 
12%. 14,15 No physiological mechanism responsible for the persistence of fatigue and 
related symptoms has yet been determined. Despite the ambiguities and controversies of 
ME/CFS, the illness experience is very much “real”. ME/CFS is an illness of physical 
and emotional disablement, with dramatic ramifications on the lives of those afflicted, 
those that care about the afflicted, and the societies they live in.
1.2 DEFINITION
ME/CFS lacks a specific biomarker or a precise phenotype. Hence, it is not surprising 
that there are currently over 20 different case definitions or diagnostic criteria.16 All these 
diagnostic criteria were recently reviewed in great detail by the Institute of Medicine 
(now called the National Academy of Medicine) and published in 2015. Importantly, they 
point out that there is significant overlap between symptoms of major depression,

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fibromyalgia, and other syndromic illnesses. Hence, the report proposed that post- 
exertional malaise and neurocognitive symptoms should be considered the core 
symptoms of ME/CFS. Since fatigue may be a normal response to a stressful event, the 
diagnosis of ME/CFS typically requires that the fatigue be present for longer than six 
months, with the exception of the 2011 International Consensus Criteria which does not 
require a six month waiting period.  All the various criteria require that other 
explanations for a patient’s symptoms to be ruled out before a diagnosis of ME/CFS can 
be made, although the list of exclusionary conditions differs across the criteria. There are 
many other challenges in establishing the diagnosis. The more recent criteria have 
developed a constellation of symptoms that need to be present for the diagnosis to be 
considered. However, the mere presence of the symptoms can lead to classification of 
healthy controls as having ME/CFS, hence close attention need to be given to the severity 
and frequency of the symptoms.
1.3 ETIOLOGY
The cause of ME/CFS remains unknown, although in many cases symptoms are thought 
to be triggered by an infection or other prodromal events such as “immunization, 
anesthetics, physical trauma, exposure to environmental pollutants, chemicals and heavy 
metals, and rarely blood transfusions”.17
The IOM in its recent report researched the possible association of infections with 
ME/CFS. They concluded that while numerous infectious agents have been associated 
with ME/CFS, only Epstein Barr Virus (EBV) has been consistently associated with the 
syndrome. Their major findings related to the association with EBV are described below. 
The possibility that EBV infection can be a trigger for ME/CFS is suggested by results of 
some prospective studies.14,18-20 Several studies also found high titers of certain 
antibodies to EBV in CFS patients, including viral capsid antigen (VCA) IgG, persistent 
titers of VCA Immunoglobulin M (IgM), or the persistence of early antigen IgG, whereas 
healthy individuals who were previously infected with EBV had only VCA IgG and 
nuclear antigen IgG antibodies.21-25 Some studies, however, including a study of twins 
discordant for disease26, were unable to find this difference.27-30 The severity of the acute 
illness may be a predictor of ME/CFS. A population-based study of ME/CFS patients 
after mononucleosis infection showed severity of illness, as measured by baseline 
autonomic symptoms and days spent in bed, to be a significant predictor of ME/CFS 6 
months after the infection.31 One double-blind, placebo controlled trial showed symptom 
improvement after 6 months in patients with elevated IgG antibody titers against EBV 
and HHV-6 following treatment with valganciclovir. There were also statistically 
significant changes in monocyte and cytokine levels, suggesting that immunomodulation 
may have been a factor in their improvement. However, the number of patients studied 
was small (N = 30), there were no differences in viral antibody titers between the two 
arms, and the patients were followed for only 9 months.32 A prospective review of 106 
ME/CFS patients with elevated serum antibody titers to EBV, CMV, or HHV-6 showed 
that 75 percent responded to long-term treatment with valacyclovir and/or valganciclovir 
(mean duration = 2.4 years). A patient was categorized as a responder if the Energy Index 
Point Score effect was greater than or equal to 1.33 A single-blind, placebo-controlled 
trial found a significant increase in NK cell activity in ME/CFS patients following

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treatment with isoprinosine.34 However, another double-blind, placebo-controlled trial of 
ME/CFS patients with elevated antibodies to EBV failed to show a difference in clinical 
improvement between acyclovir-treated participants and placebo controls at 37 days 
follow-up.35 This study included a small number of patients (N = 27) and did not assess 
immune parameters. In this model, ME/CFS represents the consequence of a persistent 
infection and successful intervention would require identifying the infective agent and 
designing therapies to thwart it.
Another possibility is that there is no ongoing infection but rather the infection acts as a 
trigger for the syndrome which is mediated by the persistent immune19,36 or 
mitochondrial37 abnormalities. A wide variety of immune abnormalities have been 
described in this population, including alterations in cytokines, expansion of specific 
immunoglobulin classes, alterations in T-cell metabolism, and NK cell function 1-3  There 
is also a greater association with autoimmune diseases and the most consistent finding 
seems to be polyclonal B cell activation, as first suggested from a placebo controlled 
clinical trial using rituximab.38 In this study 30 ME/CFS patients were randomized to 
either Rituximab 500 mg/m2 or saline, given twice two weeks apart, with follow-up for 
12 months. The primary end-point, defined as effect on self-reported fatigue score 3 
months after intervention, was negative. However, patients reported improvement at 
secondary end- points. Major or moderate overall response, defined as lasting 
improvements in self- reported fatigue score during follow-up, was seen in 10 out of 15 
patients (67%) in the Rituximab group and in two out of 15 patients (13%) in the Placebo 
group (p = 0.003). Mean response duration within the follow-up period for the 10 
responders to Rituximab was 25 weeks (range 8–44). Four Rituximab patients had 
clinical response durations past the study period. General linear models for repeated 
measures of Fatigue scores during follow-up showed a significant interaction between 
time and intervention group (p = 0.018 for self-reported, and p = 0.024 for physician-
assessed), with differences between the Rituximab and Placebo groups between 6–10 
months after intervention. There were no serious adverse events. Two patients in the 
Rituximab group with pre-existing psoriasis experienced moderate psoriasis worsening. 
The authors concluded that the delayed responses starting from 2–7 months after 
Rituximab treatment, in spite of rapid B-cell depletion, suggests that ME/CFS is an 
autoimmune disease and may be consistent with the gradual elimination of 
autoantibodies preceding clinical responses.38 However, a more recent double-blind 
placebo controlled by the same authors found that rituximab treatment did not lead to 
clinical improvement over the course of twelve months.39 In this model, a successful 
intervention could target a specific immune or metabolic deficit whose restoration would 
ameliorate symptoms.
A third possibility is that ME/CFS a syndrome with a primarily neuropsychological 
biology. ME/CFS shares many features with other syndromic diagnoses, including 
demographics, correlations with psychiatric co-morbidity, the types of symptoms 
experienced, discordance between clinical observation and subjective experience, and 
limited impact on mortality. Viewed this way, ME/CFS may reflect the somatoform 
consequence of infection that is analogous to the post-injury pain of whiplash and post- 
traumatic fibromyalgia, the headaches and dyscognition of post-concussive syndrome and 
mild traumatic brain injury, the fatigue and dyscognition following cancer therapies, the 
post-Lyme disease syndrome following infections with Lyme disease, the event-

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specific triggers of functional movement disorders, and perhaps the emerging problem of 
“long haul” COVID-19.4 Alterations in immune function, neuroendocrine performance, 
and other biological parameters would represent epiphenomenon in this model, objective 
evidence of system-wide performance changes that occur with perceived distress.39,40 
Interventions targeting a single physiological system could temporarily improve 
symptoms related to bidirectional communication with, and influence on, neural systems. 
However, such interventions would be unlikely to lead to self-evident and long-lasting 
benefit due to our essential tendencies to acclimate to chronic physiological changes and 
to develop collateral systems to circumvent dysfunction occurring in any single 
physiological mechanism. Successful interventions in the model would require dissecting 
the “master” regulatory signals and develop interventions that would downregulate all of 
the altered physiological systems in synchrony.
However, it is probable that the cause of symptoms throughout the whole population of 
persons diagnosed with ME/CFS is not homogeneous. It is possible that all three of the 
aforementioned possibilities are correct, but only for subpopulations of ME/CFS 
sufferers. These possibilities may not be relevant to persons who credit the onset of 
ME/CFS with non-infectious exposures and life stressors nor those with substantial 
premorbid psychiatric illness. A host of unusual or poorly described medical phenomena, 
such as mast cell activation disorder, Chiari malformation, myotonic muscular dystrophy, 
paraneoplastic phenomenon that can accompany occult neoplasms, and alterations in the 
microbiome, may also contribute to the development of ME/CFS symptoms in certain 
individuals. For the purpose of reducing heterogeneity in the current study, we will only 
enroll patients who have a clinical history of infection followed by chronic fatigue and 
post-exertional malaise. We have introduced the term, post-infectious ME/CFS (PI- 
ME/CFS) to refer to these patients.
1.4 EPIDEMIOLOGY
An estimated 84 to 91 percent of patients affected by the condition are not yet 
diagnosed,41 and people with ME/CFS often struggle with their illness for years before 
receiving a diagnosis. In multiple surveys, 67 to 77 percent of patients reported that it 
took longer than 1 year to receive a diagnosis, and about 29 percent reported that it took 
longer than 5 years.
1.5 DIAGNOSTIC CHALLENGES
Seeking and receiving a diagnosis can be a frustrating process for patients with ME/CFS 
for several reasons, including a lack of understanding of diagnosis and treatment of the 
condition among health care providers and skepticism about whether it is in fact a true 
medical condition. Less than one-third of medical schools include ME/CFS-specific 
information in their curriculum 42, and only 40 percent of medical textbooks include 
information on the condition 43. Some studies on awareness of ME/CFS have found high 
awareness among health care providers, but many providers believe it is a 
psychiatric/psychological illness or at least has a psychiatric/psychological 
component.44,45 ME/CFS often is seen as a diagnosis of exclusion, which also can lead to 
delays in diagnosis or misdiagnosis of a psychological problem. 46,47 Patients often

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struggle with their illness for years before receiving a diagnosis. Hence there is a need for 
a faster and more accurate diagnosis of this syndrome.
1.6 SOCIO-ECONOMIC BURDEN
Once diagnosed, moreover, many people with ME/CFS report being subject to hostile 
attitudes from their health care providers 48, as well as to treatment strategies that 
exacerbate their symptoms. 49
ME/CFS can cause significant impairment and disability that have negative economic 
consequences at the individual and societal levels. At least one-quarter of ME/CFS 
patients are house- or bedbound at some point in their lives.50 The direct and indirect 
economic costs of ME/CFS to society are estimated to be between $17 and $24 billion 
annually,51 $9.1 billion of which can be attributed to lost household and labor force 
productivity.52 Together, high medical costs and reduced earning capacity often have 
devastating effects on patients’ financial situations.52
1.7 DISABILITY
Several ME/CFS symptoms—including fatigue, cognitive dysfunction, pain, sleep 
disturbance, post-exertional malaise, and secondary depression or anxiety—may 
contribute to impairment or disability 53,54. Patients with ME/CFS have been found to be 
more functionally impaired than those with other disabling illnesses, including type 2 
diabetes mellitus, congestive heart failure, hypertension, depression, multiple sclerosis, 
and end-stage renal disease 44,55. Symptoms can be severe enough to preclude patients 
from completing everyday tasks, and 25-29% of patients report being house- or bedbound 
by their symptoms. Many patients feel unable to meet their family responsibilities and 
report having to reduce their social activities. However, these data include only patients 
who were counted in clinics or research studies and may underrepresent the extent of the 
problem by excluding those who are undiagnosed or unable to access health care.56,57
1.8 MORTALITY
Literature on mortality associated with ME/CFS is sparse. One study found that cancer, 
heart disease, and suicide are the most common causes of death among those diagnosed 
with ME/CFS, and people with ME/CFS die from these causes at younger ages than 
others in the general population. However, the authors note that these results cannot be 
generalized to the overall population of ME/CFS patients because of the methodological 
limitations of the study 58.
2
Study Objectives
2.1 Primary objective
To explore the clinical and biological phenotypes of post-infectious chronic fatigue 
syndrome (PI-ME/CFS)

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2.2 Secondary objective
To explore the pathophysiologies of fatigue and post-exertional malaise (PEM). Fatigue 
will be explored using tasks designed to create muscular and cognitive fatigue. PEM will 
be explored using an exercise stress and measuring the symptomatic and biological 
alterations that occur before and afterwards.
3
Subjects
3.1 Description of study populations
Up to 346 persons will be enrolled as part of this protocol. Up to 150 persons aged 18-60 
will be part of 3 study groups: 50 PI-ME/CFS patients, 50 healthy volunteers (HV), and 
50 asymptomatic and healthy persons with a documented history of COVID-19 infection 
(COVID-19 HV). The study has a target of completing all study procedures on 20 
enrolled participants in each group. Any participant who withdraws prior to the 
completion of the study procedures may be replaced. Up to an additional 176 persons 
reporting a community diagnosis of ME/CFS will be enrolled into focus groups to 
discuss the experience of post-exertional malaise. Up to an additional 10 healthy 
volunteers and 10 ME/CFS participants may be enrolled to refine the protocol’s 
electrophysiological and neuroimaging techniques.
The PI-ME/CFS group will consist of persons reporting persistent and severe fatigue and 
post-exertional malaise as the consequence of an acute infection without a prior history of 
medical or psychiatric illness. PI-ME/CFS pre-screening will review medical records and 
will seek to identify persons with a community diagnosis of ME/CFS that started within 6 
months after an infectious illness that do not have other potential medical causes for their 
symptoms.
Potential PI-ME/CFS participants that meet inclusion and exclusion criteria for a 
phenotyping visit on pre-screening will be invited to participate and undergo a full week 
of medical interviews and research measurements. This collected information will be 
used in a case adjudication process to determine if a participant meets criterion to return 
for a separate exercise stress visit. Exercise stress visit participants are required to meet 
more stringent inclusion and exclusion criteria to best ensure that participants have 
ME/CFS that started after an infection, meet published diagnostic criteria, have an 
appropriate level of symptom severity and physical function to undergo the protocol’s 
exercise stress procedure, and do not have exclusionary co-morbid health issues. This 
process has necessitated two sets of inclusion criteria for PI-ME/CFS participants: 
phenotyping criteria (Sections 3.2.2 and 3.3.2) and exercise stress visit criteria (Sections 
3.2.3 and 3.3.3).
All PI-ME/CFS phenotyping participants will have a ME/CFS diagnosis from their health 
care providers and, therefore, represent the illness as it is considered in the community.
For this reason, PI-ME/CFS participants will undergo all of the phenotyping procedures 
even if information that would exclude the participant from the study is discovered 
during the visit. Data collected from PI-ME/CFS participants that fail to meet exercise 
stress criterion will be used in comparative analyses and serve as a control group for 
comparisons to PI-ME/CFS participants that qualify for the exercise stress visit.

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Two other control groups have also been chosen for comparisons.
The healthy volunteer (HV) group will consist of demographically matched persons 
without clinical fatigue, free from medical disease, and presumed to have a properly 
functioning immune system. The HV group represents a standard control population for 
comparison to the PI-ME/CFS group. HV participants will take part in both the 
phenotyping and exercise stress visits.
Asymptomatic persons with a history of COVID-19 infections and a full recovery is a 
salient comparator group to PI-ME/CFS. Similar to PI-ME/CFS, COVID-19 has been 
implicated in causing a syndrome of fatigue, pain, and neurocognitive difficulties, 
currently referred to as “long haul COVID”.4-6  The COVID-19 HV group will consist of 
healthy persons who had a laboratory documented COVID-19 infection within the last 5 
years and currently do not have clinically significant fatigue and PEM. The COVID-19 
HV control group will allow for biological amongst persons with an infectious process 
that did not progress to chronic debility. COVID-19 HV participants will take part in the 
phenotyping visit but not the exercise stress visit of the protocol.
3.2 Inclusion criteria
3.2.1 Inclusion criteria for all participants
1. Adult participants aged 18-60 years at the time of enrollment.
2. Self-reported completion of at least the 7th grade of school.
3. Ability to speak, read, and understand English.
4. Willing and able to complete all study procedures
5. Participant has a primary care physician at the time of enrollment.
6. Able to provide informed consent.
3.2.2 Additional inclusion criteria for participants with PI-ME/CFS for the 
phenotyping visit
1. A self-reported illness narrative of the development of persistent fatigue and post- 
exertional malaise as the consequence of an acute infection. The persistent fatigue 
may have an acute onset or become progressively worse over 6 months.
2. Licensed Independent Practitioner documentation of ME/CFS onset:

Medical documentation of absence of symptoms prior to ME/CFS onset. 
This may include medical records, letters, or information gathered from 
telephone calls with study personnel.

Documentation of a medical evaluation for symptoms of an acute infection 
or documentation of a medical evaluation of persistent symptoms within 2 
months following an assumed infection.
3. Persistent fatigue and PEM onset less than 5 years prior to enrollment.

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3.2.3 Additional inclusion criteria for participants with PI-ME/CFS for the 
exercise stress visit
1. Be unanimously considered to be a case of PI-ME/CFS by the protocol’s 
adjudication committee.
2. Meet the 1994 Fukuda Criteria OR the 2003 Canadian Consensus Criteria for 
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome OR the Institute of Medicine 
Diagnostic Criteria.61-63
3. Have moderate to severe clinical symptom severity:

Severe fatigue as determined using the Multidimensional Fatigue Inventory 
(MFI): score of ≥ 13 on the general fatigue subscale or ≥10 on the reduced 
activity subscale.

Functional impairment as determined using the Short-Form 36 (SF-36): score 
of ≤70 physical function subscale, or ≤50 on role physical subscale, or ≤75 on 
social function subscale
3.2.4 Additional inclusion criteria for healthy volunteer group
None
3.2.5 Additional inclusion criteria for COVID-19 Healthy 
Volunteers
Documented prior COVID-19 infection as evidenced by:
1. A history of clinical manifestations compatible with COVID-19 
2. Laboratory evidence of COVID-19 infection: 
 Detection of SARS-CoV-2 RNA or antigen in nasopharyngeal swab, sputum, other sample 
source with Emergency Use Authorization/approval from the FDA; OR 
 A positive antibody test using an assay that has received Emergency Use 
Authorization/approval from the FDA
3. Has been recovered from the COVID-19 infection for at least six months and no more than 
five years. 

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3.3 Exclusion criteria
3.3.1 Exclusion criteria for all participants
1. Current or past psychotic disorder including depression with psychosis, bipolar 
disorder, and schizophrenia
2. Current DSM-5-defined major depression disorder, generalized anxiety disorder, 
post-traumatic stress disorder, panic disorder, or obsessive-compulsive disorder 
unless managed for more than six months with a stable treatment regimen
3. Current or substance use disorder within last 5 years as diagnosed on the 
Structured Clinical Interview for DSM-5 (SCID-5).
4. Current suicidal ideation
5. History of head injury with loss of consciousness or amnesia lasting greater than a 
few seconds within last five years or lasting greater than 5 minutes at any point 
during their lifetime. Persons with medical record evidence of post-concussive 
symptoms lasting more than six months are also excluded.
6. Women who are pregnant, actively seeking to become pregnant, or have been 
pregnant in the year prior to study enrollment.
7. Current or previous malignancy. Certain dermatologic malignancies (e.g. basal 
cell carcinoma) will be allowed. A history of malignancy that have fully resolved 
with surgical resection only (i.e. no chemotherapy, radiation therapy, or 
immunotherapy) will be allowed.
8. Current systemic immunologic disorders (e.g. Type 1 diabetes, rheumatoid 
arthritis) will be excluded. Allergies requiring anti-histamines may not be an 
exclusion, but allergies requiring immunosuppressants may be an exclusion.
9. Current or previous long term immune suppressive or immunomodulatory 
therapy. Systemic steroid use, even short-term, must not have been used within 
the month prior to enrollment
10. Any medical condition (eg, congestive heart failure, coronary artery disease, 
chronic obstructive pulmonary disease, severe osteoarthritis, poorly controlled 
asthma) that would make the study procedures risky for the participant (e.g. 
exercise-induced angina and asthma) or that may confound the study results (e.g. 
untreated obstructive sleep apnea, severe osteoarthritis).
11. Participation in a clinical protocol (e.g. anti-inflammatory drug intervention 
study) which includes an intervention that may affect the results of the current 
study.
12. Inability to perform the bicycling exercise task.
13. Clinically significant claustrophobia
14. Not willing to allow for research samples to be shared with other researchers.
15. Employees or staff at NIH that are directly supervised by the primary investigator 
or associate investigators.
3.3.2 Additional exclusion criteria for participants with PI-ME/CFS for 
phenotyping visit
1. Significant neurological disorder (e.g. neurodegenerative disorder, stroke, 
epilepsy).

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2. PI-ME/CFS disease severity that makes it impossible for the volunteer to leave 
the home or requires inpatient treatment.
3. Suspected, probable, or confirmed Lyme disease per 2011 CDC Lyme Disease 
National Surveillance Case Definitions.
4. Underlying illness that may cause fatigue such as thyroid dysfunction, hepatitis, 
or other systemic diseases.
3.3.3 Additional exclusion criteria for participants with PI-ME/CFS for exercise 
stress visit
1. Current (within 1 week) use of prescription or over-the-counter medications, herbal 
supplements, or nutraceuticals that may influence brain excitability that the potential 
participant is either unwilling or clinically unable to safely wean off for the duration of 
the period of the exercise stress visit. The possibility for a potential participant to be 
weaned off medication will be cooperatively determined by both the clinical investigative 
team and personal physicians. Examples of medications that influence brain excitability 
include tricyclic antidepressants, hypnotic, antiepileptic, antipsychotic medication, 
stimulants, antihistamines, muscle relaxants, dopaminergic medications, and sleep 
medications.
3.3.4 Additional exclusion criteria for healthy volunteer group
1. Substantial daily fatigue as determined using PROMIS-SF Fatigue: score of > 17.
2. Significant neurological disorder (e.g. neurodegenerative disorder, stroke, 
epilepsy).
3. Current (within 1 week) use of prescription or over-the-counter medications, 
herbal supplements, or nutraceuticals that may influence brain excitability
3.3.5 Additional exclusion criteria for COVID-19 healthy volunteer 
group
1. Substantial daily fatigue as determined using PROMIS-SF Fatigue: score of > 17
2. Significant neurological disorder (e.g. neurodegenerative disorder, stroke, 
epilepsy).
3. Current (within 1 week) use of prescription or over-the-counter medications, 
herbal supplements, or nutraceuticals that may influence brain excitability
4
Study Design and Methods
4.1 Study overview
This is a single center, cross-sectional, phase I pilot study to explore the clinical and 
biological phenotype of PI-ME/CFS in order to deeply characterize the syndrome and 
compare it to relevant control conditions. Biological specimens will also be collected for 
the development of neuronal and animal models of PI-ME/CFS. Study procedures will 
include an initial phenotyping visit, where clinical and biological information will be 
collected. The collected information from the phenotyping visit for PI-ME/CFS 
participants will be used in a case adjudication process utilizing an expert diagnostic 
committee to determine case validity. PI-ME/CFS participants adjudicated to be valid 
cases, meet recognized diagnostic and symptom severity criteria, and are able to taper off

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medications that alter brain excitability will return to the NIH Clinical Center for an 
exercise stress visit. The results from participants with PI-ME/CFS will be compared 
against results from two distinct control groups: healthy volunteers and COVID-19 HVs. 
Prior to conducting exercise stress visits, the investigative team will establish and conduct 
up to four telephone focus groups to gather information for use in developing the 
qualitative PEM interview guide used during the exercise stress visit. Up to an additional 
20 focus groups will be conducted to expand on the information collected in these initial 
focus groups. The purpose of this data collection would be to provide more extensive 
information about the similarities and differences of PEM in daily life compared with that 
occurring after CPET. Additionally, a small number of healthy volunteers and ME/CFS 
patients will be enrolled in technical development sub-study that will be used to refine 
the protocol’s electrophysiological and neuroimaging techniques used during the exercise 
stress visit.
It is anticipated that most participants in both the PI-ME/CFS group and the healthy 
volunteer group will not be currently enrolled on other NIH protocols at the time of study 
participation. Participants from the COVID-19 HV group may be co-enrolled in NIH 
protocols or may be self-referred. Focus group participants are not anticipated to be 
currently enrolled in other NIH protocols. Participation in the technical development sub-
study will be made available to all healthy volunteers inquiring about participation in the 
protocol.
Healthy volunteers in the technical development sub-study may also participate in the 
phenotyping and exercise stress visits. Participation in the technical development sub- 
study will be made available to documented ME/CFS patients that inquire about 
participation in the protocol and would otherwise be eligible to participate but do not 
meet study criteria for post-infectious causation or for having symptoms for less than 5 
years. ME/CFS patients that participate in the technical development sub-study are 
excluded from participation in the phenotyping and exercise stress visits but may 
participate in focus groups.
The phenotyping visit (Visit 1) is designed to clearly define and document the 
characteristics of the study populations as well as collect biological samples. Eligible 
participants will undergo a set of research measurements and procedures. The results 
from the phenotyping visit will enable cross-sectional comparisons between the three 
study groups.
The exercise stress visit (Visit 2) will occur up to 12 months after the phenotyping visit. 
The exercise stress visit is designed around an exercise intervention designed to evoke 
the characteristic post-exercise malaise of PI-ME/CFS. All participants will undergo 
careful clinical description and a variety of biological measurements before, during, and 
after peak exercise testing. Qualitative interviews, subjective reporting on questionnaires, 
physiological measurements, neuroimaging, and biological samples will be serially 
collected. Only HV and PI-ME/CFS participants will be invited to participate in the 
exercise stress visit. These measurements will enable cross-sectional comparisons 
between these two study groups as well as comparisons between pre- and post-exercise 
state within and between study groups.

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Participants in this study will undergo a great number of procedures in a relatively short 
period of time. All of the procedures that are to be performed in this study are standard 
research measurements, with well-defined risks and discomforts. No particular 
measurement or procedure is exceedingly burdensome, however the cumulative burden 
of undergoing all of these procedures is significant. This burden is a reasonable one, as it 
is the intent of this study to induce fatigue and PEM in its participants. However, PI- 
ME/CFS patients are defined by their vulnerability to exhaustion. It is probable that some 
participants will be unable complete the entire study. Some participants may also be so 
exhausted by the study that they may require some time to recover prior to travelling 
home. Thus, the study team will be solicitous of the participants in regard to their 
comfort and flexible to provide for their needs.
This ‘Phase I’ study will analyze the collected data in an exploratory manner. The goal of 
these analyses is to identify physiological alterations for the purpose of hypothesis 
generation. A broad array of laboratory methods that query different aspects of human 
physiology will be performed on collected samples. The protocol is looking for positive 
results that would generate testable hypotheses and for negative results that will provide a 
sense of the biological measurements and physiological mechanisms where large 
differences are unlikely to be seen with future study. De-identified data and biological 
samples will be shared with a wide range of collaborators, both within NIH and across 
the world. A larger ‘Phase II’ study is planned that will use these results to determine 
potential mechanisms for intervention and estimate effect sizes for larger clinical and 
interventional studies.
4.2 Recruitment
Recruitment for the study will vary by group.

PI-ME/CFS patients may be referred to this study from the CDC CFS Multi-site 
Clinical Study. This ongoing natural history study is funded to assess ME/CFS 
patients until September 2017.

COVID-19 HV participants may be referred from NIH clinics who study these 
conditions, in particular the investigators involved in Protocol 000084: 
Natural History of Post-Coronavirus Disease 19 Convalescence at the 
National Institutes of Health. Participants will not be directly recruited by 
physicians with whom they have a therapeutic relationship with.

Patients from all three groups may be recruited from the Patient Recruitment and 
Public Liaison (PRPL) office and by self-referral. A study investigator will contact 
potential participants via the contact information provided by the PRPL inquiry form 
to evaluate eligibility.

Participants may also be recruited from other NIH protocols. Non-Associate 
Investigators working on other NIH protocols will be provided with IRB-approved 
recruitment flyers. If appropriate, these non-AI investigators may send flyers to their 
study participants (both healthy volunteers and patients) by mail with a cover letter to 
inform them of the study opportunity or provide flyers to study participants in person.
All potential participants will undergo a pre-screening process to assist in the 
determination of eligibility. Persons that have inquired about potential participation will

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be contacted by phone. A check-list based on the study inclusion and exclusion criteria 
will be used by the investigative team to collect information relevant to determining 
eligibility. Persons obviously not meeting eligibility criteria will be informed at this time. 
Potential PI-ME/CFS and COVID-19 HV participants will be informed that medical 
record review is one of the eligibility criteria. Study staff will work with potential 
participants in obtaining relevant medical records. Costs associated with obtaining 
medical record will be paid for by the protocol. Medical records obtained will be kept 
confidential and locked in a filing cabinet when not in use. If medical record review 
determines that a person is ineligible to participate, they will be notified. Medical records 
on ineligible persons may be kept until the completion of the study. Participants may 
request their records to be returned or destroyed at any time.
Permission to contact relevant medical care providers may also be requested. Some 
potential participants will be taking medications that would require being tapered off 
during the study, while others may need to have facilitated access to medical care after 
study visits.
Employees of the NIH may participate in this protocol. There will be no direct 
solicitation of employees or staff members by supervisors. All recruitment materials, 
including those specific for NIH, will be approved by the IRB prior to use.
Advertisements, study-specific website language, and Telephone Screening Forms are 
described in detail in Appendix 26.5, 26.6, and 26.7 respectively.
Once the potential participant is determined to be eligible for the study, a date will be set 
for the phenotyping visit. It is anticipated that 1-2 potential participants will be identified 
and pre-screened per week.
4.3 Focus Groups
4.31
Focus Group Study A
In order to gather information for use in creating a meaningful interview guide for the 
PEM qualitative interview planned during the Exercise Stress Visit, the investigative 
team will conduct up to 10 focus groups. Each group will contain between three to nine 
ME/CFS patients, with the maximum number of focus group participants being 36. Focus 
group participants will not be eligible to participant in other study procedures and 
represent their own recruiting pool. NIH employees will not be eligible to participate in 
the focus groups. The focus groups will take place over the telephone to accommodate a 
non-mobile population.
Focus group participants will be persons with ME/CFS in the community willing to 
participate in telephone discussions with the investigative team to assist in the 
development of a qualitative interview tool for eliciting the experience of PEM.
4.31.1
Inclusion criteria for focus group A participants
1. Adult 18 years or older
2. Self-report of being diagnosed with ME/CFS in the community.

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3. Self-report of having experienced post-exertional malaise.
4. Ability to speak, read, and understand English.
5. Able and willing to participate in focus group telephone call.
4.31.2
Exclusion criteria for focus group participants
1. Participation in the phenotyping and exercise stress studies.
2. Decline to be audio-taped during the focus group.
A letter of invitation (Appendix 26.4) will be sent to community advocacy groups to be 
shared with their members and may posted on-line at NIH ME/CFS websites. Persons 
interested in participating in the focus group will contact the investigative team. Potential 
focus group participants will be screened for inclusion/exclusion using a focus group 
participation checklist (Appendix 26.4). Potential focus group participants will be 
informed that focus group participation precludes participation in the phenotyping and 
exercise stress studies. The investigative team will contact interested individuals to 
answer any questions and address concerns, and schedule times for obtaining telephone 
informed consent.
Persons found to be eligible will be mailed a copy of the focus group consent form 
(Appendix 26.4). The individual will be instructed to read the consent form and be ready 
at the appointed date and time to review the consent document with an investigator 
authorized to obtain consent. Decision making capacity will be assessed and 
documented. If the subject is determined not to have decision making capacity, the 
telephone consent process will be halted, and the participant will be determined ineligible 
for participation in the focus group.
Potential participants with decision making capacity will be asked: "Do you agree to 
voluntarily participate in this study?" The consenting investigator will document the date 
and time oral consent was obtained on a consent procedure note. The participant will sign 
and date the consent form in the presence of a witness. The witness will also sign and 
date the consent form. The subject will be instructed to mail the consent form back to the 
site, where the investigator will sign and date the form. The investigator will then send a 
copy of the form with all signatures to the subject. The original consent document will be 
placed in the research chart.
After the investigative team receives the informed consent form, the focus group 
participant will be contacted to schedule a focus group teleconference time.
The purpose of the focus groups is to gather information to create the interview protocol 
to be used for the semi-structured interviews. Each focus group will be a joint discussion 
in which all of the scheduled participants will participate together. An overview guide to 
the focus group discussion has been developed (Appendix 26.4). Barbara Stussman will 
moderate the focus groups. Persons who decline to be audio-taped will not be included in 
the focus groups. Focus groups will be rescheduled if less than three participants make 
themselves available for the telephone call.

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Recruitment for the focus groups will seek to categorize participants by level of 
disability: persons with symptoms that have maintained their activity level, persons 
whose activity has been limited by symptoms, and persons fully disabled and/or 
housebound from their symptoms.
Data collected during focus groups will consist of audio recordings which will be sent to 
a professional service for transcription via a secure website. Data, recordings, and 
transcriptions will be stored in locked cabinets. Transcriptions will NOT contain any 
identifying information and will be labeled with a number only. Basic demographics will 
be collected on all focus group participants but will not be associated with the 
corresponding transcriptions and will be kept secure and confidential. Demographics to 
be collected from focus group participants include: (sex, age (18-29; 30-39; 40-49; 50-59; 
60+), race (Asian, Black, White, Other), ethnicity (Hispanic; non-Hispanic), years of 
diagnosis of CFS (0-4; 5-9; 10-14; 15-19; 20+). Contact information that contains 
personally identifying information for focus group participants will be kept on file for 
five years after the conclusion of the study.
The risks of focus group participation include potential breach of confidentiality and 
emotional feelings that may arise with discussing personal feelings in a group setting. 
There is no direct benefit from focus group participation. Focus group participants will 
not be compensated for their participation.
4.32
Focus Group Study B
As of October 2018, the results of the initial focus groups (Focus Group Study A) have 
been used to create the interview guide being used during the exercise stress visit. The 
initial qualitative analysis of the collected data provides a description of post-exertional 
malaise in which there may be four key symptom concepts (cognitive difficulties, 
extreme fatigue, flu-like symptoms, and muscle pain/weakness) and a time course in 
which to expect the onset of PEM. The medical literature has a paucity of detailed 
qualitative studies about PEM in ME/CFS; there is currently only a single study of 19 
persons.64 An expansion of the work performed in Focus Group Study A work has the 
potential to contribute to broader knowledge of PEM.
Focus group Study B will expand the scope of our focus group study activities. This 
expansion will include collecting more extensive demographic information, information 
about symptom severity, information about CPET experiences, and collection of medical 
records for diagnostic confirmation purposes. The purpose of this data collection would 
be to provide more extensive information about the similarities and differences of PEM 
in daily life compared with that occurring after CPET.
For Focus Group Study B, the investigative team will conduct up to 20 focus groups. 
Each group will contain between three to seven ME/CFS patients, with the maximum 
number of focus group study B participants being 140. Focus group participants will not 
be eligible to participant in the main study (Visit 1 phenotyping and Visit 2 exercise 
stress) but may participate in the Technical Development Substudy (TDS) and represent 
their own recruiting pool. NIH employees will not be eligible to participate in the focus

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groups. The focus groups will take place over the telephone to accommodate a non- 
mobile and geographically dispersed population.
Focus group participants will be persons with ME/CFS in the community willing to 
participate in telephone discussions with the investigative team to describe their 
experiences of PEM and provide medical records for diagnostic confirmation.
Inclusion criteria for focus group B participants
1. Adult 18 years or older
2. Self-report of being diagnosed with ME/CFS in the community.
3. Self-report of having experienced post-exertional malaise.
4. Ability to speak, read, and understand English.
5. Able and willing to participate in focus group telephone call.
6. Willing to share their medical records with the investigative team for diagnostic 
confirmation.
Exclusion criteria for focus group B participants
1. Participation in the phenotyping and exercise stress studies.
2. Decline to be audio-taped during the focus group.
Initially, participants will be recruited from persons who have contacted the investigative 
team about participating in any aspect of this protocol. These potential focus group 
participants will be contacted by phone and screened for inclusion/exclusion using Focus 
Group B Participation Checklist (Appendix 26.4). Potential focus group participants will 
be informed that focus group participation precludes participation in the phenotyping and 
exercise stress studies. The investigative team will contact interested individuals to 
answer any questions and address concerns, work with participants in completing 
paperwork to obtain pertinent medical records, and schedule times for obtaining 
telephone informed consent. The NIH will cover the costs of obtaining medical records. 
If recruitment goals are not met using the protocol’s recruitment pool, a letter of 
invitation (Appendix 26.4) will be sent to community advocacy groups to be shared with 
their members and may posted on-line at NIH ME/CFS websites. Persons interested in 
participating in the focus group will contact the investigative team and undergo screening 
as detailed above. The investigative team may target specific under-represented ME/CFS 
sub-groups, such as minorities with ME/CFS, males with ME/CFS, persons aged 18-29 
with ME/CFS, and bedbound ME/CFS participants.
Persons found to be eligible will be mailed a copy of the focus group consent form 
(Appendix 26.4). The individual will be instructed to read the consent form and be ready 
at the appointed date and time to review the consent document with an investigator 
authorized to obtain consent. Decision making capacity will be assessed and 
documented. If the subject is determined not to have decision making capacity, the 
telephone consent process will be halted, and the participant will be determined ineligible 
for participation in the focus group.

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Potential participants with decision making capacity will be asked: "Do you agree to 
voluntarily participate in this study?" The consenting investigator will document the date 
and time oral consent was obtained on a consent procedure note. The participant will sign 
and date the consent form in the presence of a witness. The witness will also sign and 
date the consent form. The subject will be instructed to mail the consent form back to the 
site, where the investigator will sign and date the form. The investigator will then send a 
copy of the form with all signatures to the subject. The original consent document will be 
placed in the research chart.
After the investigative team receives the informed consent form, the focus group 
participant will be contacted to schedule a focus group teleconference time and to 
coordinate the collection of medical records.
The purpose of the focus groups is to gather information to collect information about the 
experience of PEM in daily life and the experience of PEM after CPET. Each focus 
group will be a joint discussion in which all of the scheduled participants will participate 
together. Overview guides for the Focus Group Study B discussions have been developed 
(Appendix 26.4). Barbara Stussman will moderate the focus groups. Persons who decline 
to be audio-taped will not be included in the focus groups. Focus groups will be 
rescheduled if less than three participants make themselves available for the telephone 
call.
Recruitment for the focus groups will seek to categorize participants by level of 
disability: persons with symptoms that have maintained their activity level, persons 
whose activity has been limited by symptoms, and persons fully disabled and/or 
housebound from their symptoms.
Data collected during focus groups will consist of audio recordings which will be sent to 
a professional service for transcription via a secure website. Data, recordings, and 
transcriptions will be stored in locked cabinets. Transcriptions will NOT contain any 
identifying information and will be labeled with a number only. Basic demographics will 
be collected on all focus group participants but will not be associated with the 
corresponding transcriptions and will be kept secure and confidential. The demographic 
and descriptive information that will be collected can be reviewed in Focus Group B 
Participation Checklist (Appendix 26.4). Contact information that contains personally 
identifying information for focus group participants will be kept on file for five years 
after the conclusion of the study.
The risks of focus group participation include potential breach of confidentiality and 
emotional feelings that may arise with discussing personal feelings in a group setting. 
There is no direct benefit from focus group participation. Focus group participants will 
not be compensated for their participation.
An in-depth qualitative analysis process, thematic analysis, will be used to systematically 
code and categorize the textual data. Two investigators will read over the transcripts 
several times to allow immersion into the data, and then agreed upon a preliminary

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categorical structure or “coding scheme.” They then independently “coded” all of the 
transcripts (i.e. the textual data), which involved identifying salient categories, collating 
those categories into overarching themes, and assigning names to the themes.65,66 After 
the two researchers independently coded all the textual data, codes will be compared to 
identify disagreements and meetings will be conducted to discuss coding differences at- 
length and reach consensus. A qualitative software package (such as MAXQDA) will be 
used to highlight text within the transcripts and create categories and sub-categories 
electronically.
4.4 Technical Development Sub-Study
During the Exercise Stress Visit (Visit 2), participants will undergo a sequence of 
transcranial magnetic stimulation (TMS), functional magnetic resonance imaging (fMRI), 
and electroencephalography (EEG). An overview of those neurological measurements are 
described in full in Sections 4.6.15 and 4.6.16 below. To refine these techniques, up to 10 
healthy volunteers and up to 10 ME/CFS participants will be enrolled on an outpatient 
basis for the purpose of having the TMS, fMRI, and EEG measures streamlined. This
sub-study will be used to refine the timing and sequencing of different measurements and 
tasks, to ensure that the various equipment involved works harmoniously with each other, 
provide the investigative team experience in synchronizing these different measurements 
and using them in ME/CFS volunteers.
4.4.1
Inclusion criteria for all participants in the technical development sub- 
study
1.
Adult participants aged 18-60 years at the time of enrollment.
2.
Ability to speak, read, and understand English.
3.
Willing and able to complete all study procedures
4.
Right-handed
5.
Able to provide informed consent.
4.4.2
Exclusion criteria for the technical development sub-study
1.
Active infection (e.g. influenza, urinary tract infection) by history, 
physical examination. These participants may be re-evaluated after the 
acute infection resolves.
2.
Current or past psychotic disorder including depression with psychosis, 
bipolar disorder, and schizophrenia
3.
Current DSM-5-defined major depression disorder, generalized anxiety 
disorder, post-traumatic stress disorder, panic disorder, or obsessive- 
compulsive disorder unless managed for more than six months with a 
stable treatment regimen
4.
Current substance use as determined during medical history and by urine 
toxicology. Marijuana use will not be considered an exclusion.
5.
Current suicidal ideation as determined during medical history
6.
Women who are pregnant,
7.
Current or previous malignancy. Certain dermatologic malignancies (e.g. 
basal cell carcinoma) will be allowed. A history of malignancy that have

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fully resolved with resection only (i.e. no chemotherapy, radiation therapy, 
or immunotherapy) will be allowed.
8.
Current immunologic disorder (e.g. Type 1 diabetes, rheumatoid arthritis)
9.
Current or previous long term immune suppressive or immunomodulatory 
therapy.
10.
Any medical condition that would exclude from participation in the 
phenotyping and exercise stress visits (eg, congestive heart failure, 
coronary artery disease, chronic obstructive pulmonary disease, severe 
osteoarthritis, poorly controlled asthma) or make the study procedures 
risky for the participant (e.g. seizure disorder).
11.
Participation in a clinical protocol (e.g. centrally-acting drug intervention 
study) which includes an intervention that may affect the results of the 
technical development sub-study.
12.
Clinically significant claustrophobia
13.
Not willing to allow for research samples to be shared with other 
researchers.
14.
Employees or staff at NIH that are directly supervised by the primary 
investigator or associate investigators.
4.4.3
Additional inclusion criteria for ME/CFS participants in the technical 
development study
1.
Licensed Independent Practitioner documentation of ME/CFS
4.4.4
Additional exclusion criteria for ME/CFS participants in the technical 
development study
1. Significant neurological disorder (e.g. neurodegenerative disorder, stroke, 
epilepsy).
2. ME/CFS disease severity that makes it impossible for the volunteer to leave 
the home or requires inpatient treatment.
3. Current treatment for underlying illness that may cause fatigue such as thyroid 
dysfunction, hepatitis, or other systemic diseases.
Right-handed healthy volunteers interested in participating in the protocol may also be 
invited to participate in the technical development sub-study. Qualified healthy 
volunteers may participate in both the sub-study and the phenotyping/exercise stress 
visits. For participants interested in both parts of the study, participation in the technical 
development sub-study must occur prior to the phenotyping visit (Visit 1). Healthy 
volunteers participating in the technical development sub-study will complete a specific 
informed consent form; participants in both parts of the study will need to complete two 
informed consent forms.
Participation in the technical development sub-study will be made available to right- 
handed ME/CFS patients with that inquire about participation in the protocol, are 
generally healthy, have no medical issues that would increase risk of having TMS and 
MRI, but are found not meet the protocol’s strict criteria for post-infectious causation or

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symptom duration. PI-ME/CFS participants that enroll in the phenotyping and exercise 
stress visits will be ineligible for participating in the technical development sub-study.
All participants in the technical development sub-study will have a detailed history and 
physical examination and urine toxicology. ME/CFS diagnostic status will be determined 
using the Fukuda, Canadian Consensus Criteria, and the Institute of Medicine Diagnostic 
Criteria. Participants will need to meet at least one of these criteria. Women of 
childbearing age will have urine pregnancy testing performed within 24 hours of fMRI 
and/or TMS. For participants undergoing fMRI that have not had a clinical MRI scan at 
the NIH in the past year, the investigative team will collect the clinical MRI sequences 
prior to fMRI experiments. It is anticipated that all measurements in the technical 
development sub-study, including TMS, MRI, and EEG, can be completed during 1-3 
outpatient visits.
The timing of each test in the electrophysiology and fMRI sequences is crucial for 
obtaining precise images and determining how the different measured parameters change 
following the physical and mental fatiguing tasks. Once a task begins, it will trigger a 
chain of operations. For the TMS procedure, this includes applying single and double 
TMS pulses at precise intervals during the electrophysiology sessions. For the fMRI 
procedure, this includes synchronizing the markers of the fMRI sequence with both the 
EEG and the behavioral performance during the fatiguing tasks. Normal differences 
between individuals’ task adherence can introduce a jitter in synchronization, ranging 
from differential ability to perform a task at different time points to missed trials. This 
sub-study will train the team to troubleshoot missed trials in real-time, identify potential 
weak points in the chain of events that need special attention, and adapt the procedure 
accordingly. The sub-study will also identify the average and minimum number of 
block/task that healthy volunteers and ME/CFS patients yield, which will help the 
investigators determine the minimum set of recordings that are required to extract the 
complete amount of information desired.
Participants will also complete questionnaires as part of the technical development sub- 
study. These include: NIH-BFI. MFI-20, MASQ, SF-36, PROMIS, BDI-II, BAI, and
PDS. These questionnaires are described in detail in Sections 4.5.5 and 4.6.6.
4.5 Phenotyping Visit (Visit 1: Phenotyping)
The phenotyping visit will take place at the NIH Clinical Center up to 12 months before 
the exercise stress visit and is expected to last 5 days for HV and COVID-19 HV 
participants and 10 days for PI-ME/CFS participants.  Phenotyping may occur on either 
an outpatient or an inpatient admission basis, depending on convenience. Information 
collected during the phenotyping visit will be used to determine PI-ME/CFS case status 
as well as to determine eligibility for the exercise stress visit. All procedures are for 
research purposes.
Consent will be obtained before any study procedures, including screening procedures, 
are done. Eligibility criteria will be reviewed during the consent process. An eligibility 
criteria checklist will be reviewed on Visit 1 completion and documented in the 
protocol’s research records.

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During the phenotyping visit, clinical information that is collected may impact participant 
eligibility. All participants in the PI-ME/CFS group will have documentation of being 
diagnosed with the illness in the community. In this way, PI-ME/CFS participants found 
not to meet inclusion/exclusion criteria during the phenotyping visit still represents what 
ME/CFS is in the community. For this reason, PI-ME/CFS participants that are found to 
have alterative or concomitant diagnoses that would otherwise exclude from study 
participation may still be invited to complete all of the phenotype visit procedures.
Participants in the HV and COVID-19 HV groups may be found to meet exclusion 
criteria during Visit 1. Such participants are also permitted to complete Visit 1 
procedures, as long as they do not have substantial daily fatigue nor concerns for safety. 
Samples and data from these participants will be used in the primary analysis. HV and 
COVID-19 HV participants found to have substantial daily fatigue will have their study 
participation ended immediately and will not be used in primary analyses. Study 
participation for HV individuals meeting exclusion criteria will be complete at the end of 
Visit 1. Study participation for all COVID-19 HV individuals will be complete at the end 
of Visit 1.
Some participants may be found to have clinical and/or laboratory evidence of an acute 
and reversible inflammatory reaction. Such reactions may be related to infections 
(common cold, urinary tract infections), toxic exposures (food poisoning), or allergies. If 
acute reversible inflammation is noted, participants will be provided appropriate therapy 
to speed resolution. Certain measurements will be influenced by acute reversible 
inflammation (i.e. immunological measurements); these measurements will be drawn 
after all signs of inflammation have resolved. All questionnaires and case report form 
information are detailed in Appendix 26.2 and Appendix 26.3.
During the active recruiting period of this protocol, a series of Amendments have 
expanded the optional research procedures offered during Visit 1. Also, some participants 
did not complete certain measurements because of equipment failures or staffing issues 
during their Visit 1. Participants who have completed Visit 1 prior to Amendment 11 may 
be offered the opportunity to return to NIH to undergo these additional measurements and 
any missed Visit 1 measurements. Such participants will be reconsented prior to 
participation. Travel, accommodations, and payment will be provided as detailed in 
Section 25: Travel and Compensation. There is no time limit in regard to re-contacting 
participants for returning for additional Visit 1 measurements.
The following procedures will be completed during the phenotyping visit:
4.5.1 : History and Physical Examination: A comprehensive clinical assessment will take 
approximately 2-3 hours and will include measurement of vital signs, a detailed history, 
and physical examination. Detailed information about symptoms that comprise major PI- 
ME/CFS diagnostic criteria will be collected.61,62,67 Additional time is anticipated to be 
necessary for the PI-ME/CFS group. Participants will be seen by a LIP each day they are 
in the hospital. Progress notes will be documented in CRIS.

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4.5.2 : Intravenous line placement: An intravenous line will be placed. This will allow for 
obtaining serial blood samples, administration of gadolinium, and cytapheresis. IV 
placement typically takes between 5-30 minutes.
4.5.3 : Blood collection: The following tests will be done at the NIH Clinical Center 
laboratory to determine study eligibility:
a.
Acute care panel1
b. Mineral panel
c.
Hepatic panel
d. Complete blood count with differential
e.
Prothrombin time (PT), international normalized ratio (INR), and partial 
thromboplastin time (PTT)2
f.
Thyroid stimulating hormone (TSH), free thyroxine (T4), triiodothyronine 
(T3)
g. Iron, ferritin, transferrin saturation
h. Lipid panel1
i.
Hemoglobin A1c
j.
Anti-nuclear antibody (ANA), Rheumatoid factor (RH), anti-cyclic 
citrullinated antibody (anti-CCP), anti-Smith antibody, anti-RNP, ssA, and 
ssB2
k. Vitamin B12, 25(OH) Vitamin D, 1,25(OH)2 Vitamin D, folate2
l.
Creatine Kinase
m. C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and d-dimer
n. Quantitative immunoglobulins
o. Flow cytometry for lymphocyte subsets
p. Human immunodeficiency virus (HIV) by enzyme-linked immunosorbent 
assay (ELISA)2
q. PCR for EBV and CMV
r.
Antibodies to C6 peptide
s.
Hepatitis panel2
t.
Rapid plasma reagin (RPR)2
u. Serum tryptase level
v. Heavy metal screening
w. Serum pregnancy testing (may be performed for women of childbearing 
potential if problems arise with obtaining urine pregnancy testing).
x. Additional blood testing may be needed if clinically indicated to investigate 
for underlying systemic illnesses or other causes of fatigue.
1Fasting labs
2Results within 12 weeks of screening may be used in lieu of these tests
Additional blood may be drawn for use in live cell research measurements and/or 
stored for future use. A blood draw will take about 30 minutes. No more than 150 ml 
of blood will be drawn in a single day during Visit 1. No more than 550mL of blood 
will be drawn within an 8-week period.

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4.5.4 : Urine collection: Urine will be collected for urine toxicology screening and may be 
used to test for pregnancy in of women of childbearing potential. A sample of urine will 
also be collected and stored for future use. Urine collection takes up to 5 minutes.
4.5.5 : Symptoms Assessment: Symptom and health questionnaires will be administered. 
Some of these instruments will be scored and used to determine symptom severity as 
detailed in the exercise stress visit inclusion/exclusion criteria.
a.
Short-Form 36 (SF-36): The SF-36 is easily administered and reliably reflects 
health-related quality of life outcomes. This questionnaire has 36-items that 
assess eight health issues: physical functioning, bodily pain, role limitations 
due to physical health problems, role limitations due to personal or emotional 
problems, general mental health, social functioning, energy/fatigue, and 
general health perceptions. The SF-36 has been tested and validated 
extensively in a number of clinical populations and was developed for self- 
administration by patients. This questionnaire is part of the PI-ME/CFS 
research definition (7-10 minutes).68
b. CDC Symptom Inventory (CDC-SI): The CDC-SI will be used to collect 
information on occurrence, frequency, and intensity of symptoms common in 
ME/CFS and other fatiguing illnesses. (5-10 minutes).69
c.
Multidimensional Fatigue Inventory (MFI): The MFI is a validated 20-item 
self-report instrument designed to measure fatigue severity.70 The items are 
designed to assess general, physical, emotional, and mental manifestations of 
fatigue as well as vigor, an estimate of the patient’s energy level. The MFI-20 
has been validated in chronic fatigue syndrome patients.71 This questionnaire 
is part of the PI-ME/CFS research definition (10 minutes).
d. Patient Reported Outcomes Measurement Information System – Short Forms 
(PROMIS-SF) PROMIS is a system of highly reliable, precise measures of 
patient–reported health status for physical, mental, and social wellbeing. 
These computer adaptive tests are generally less than 6 questions and 
developed from more than 1000 datasets from multiple disease populations 
including cancer, heart disease, rheumatoid and osteoarthritis, psychiatric 
conditions, spinal cord injury, and chronic obstructive pulmonary disease. 
Initial psychometric properties showed internal consistency reliability 
coefficient of 0.81. PROMIS-SF include Fatigue, Pain Behavior, Pain 
Interference, Pain Intensity, Global Health, Emotional Distress – Anxiety, 
Emotional Distress- Depression, Sleep-Related Impairment and Sleep 
Disturbances (10 minutes).72
e.
The National Institutes of Health – Brief Fatigue Inventory (NIH-BFI) is a 7- 
item clinician-administered questionnaire. To assess reliability, a principle 
component analysis was applied and yielded a single component. Further, the 
NIH-BFI produced Cronbach alphas ranging from .81-.88. The NIH-BFI is

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strongly correlated with fatigue items from validated clinician-administered 
depression and mania scales. (2 minutes).73
f.
The McGill Pain Questionnaire (MPQ): a list of 20 groups of adjectives to 
describe sensory, affective and evaluative aspects of pain. Using the MPQ, 
participants will be asked to describe their pain experience (2 minutes).74
g. The Neuropathic Pain Scale (NPS): a questionnaire designed to assess distinct 
qualities associated with neuropathic pain. It measures both the quality and the 
intensity of the neuropathic sensations. (5 minutes).75
h. Polysymptomatic Distress Scale: Polysymptomatic Distress Scale (PSD): The 
polysymptomatic distress scale is a self-administered instrument that 
determines both the distribution of painful areas across the body and an 
estimate of related symptom burden. Evidence supports the reliability and 
validity of the PSD in the general population (5 minutes).76
i.
Patient Health Questionnaire-15 (PHQ-15): A validated questionnaire used to 
assess somatic symptom severity and the potential presence of somatization 
and somatoform disorders (3-5 minutes).77
j.
Pittsburgh Sleep Quality Index (PSQI): The PSQI measures sleep quality over 
a 1-month period with 19 questions in 7 clinically-derived component scores. 
This instrument has a sensitivity of 89.6% and a specificity of 86.5% for 
identifying a sleep disorder (PSQI<5) in a clinical sample (15 minutes).78
k. Fatigue Catastrophizing Scale is a 10-item paper/pencil questionnaire to 
measure catastrophizing related to the fatigue experience. This questionnaire 
takes less than two minutes to complete. It uses a 5-point rating scale (1= 
never true) to (5 = all the time true) with proven high internal consistency 
reliability (coefficient alpha = 0.85 – 0.92) (2 minutes).79
l.
The Multiple Ability Self-Report Questionnaire (MASQ): A 38-item 
questionnaire that assesses the subjective appraisal of cognitive difficulties in 
five cognitive domains: language, visual-perceptual ability, verbal memory, 
visual-spatial memory, and attention/concentration. The MASQ sub-scales are 
scored on an 8–40 point scale with high scores indicating greater perceived 
difficulties (10 minutes).80
m. Belief about Emotions scale: A validated questionnaire designed to measure 
the beliefs of expressing negative thoughts and feelings (3-5 minutes).81
4.5.6 : Psychological Assessment: A licensed mental health professional will perform 
psychological evaluation and the Structured Clinical Interview for DSM-5 (SCID-5) 
(approximately 2 hours). Psychological assessment will be optional for the DLI-A group.

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4.5.7 : Psychological inventories
a.
Psychological inventories will be administered. These may include: 
Composite International Diagnostic Interview Trauma Section (CIDI- 
Trauma): A validated survey that characterizes a participant’s previous 
traumatic experiences. The CIDI-Trauma quantifies and evaluates the number 
of adverse events experienced by subject, and relates these to a host of 
possible disorders under the umbrella of the DSM-IV diagnostic manual (10- 
15 minutes).82
b. Post-traumatic Stress Diagnostic Scale (PDS): A validated instrument for the 
epidemiologic diagnosis of Post-traumatic Stress Disorder (10 minutes).83
c.
Childhood Trauma Questionnaire Short Form (CTQ-SF): A validated 
instrument that characterizes potential traumatic life experiences in early 
childhood (10 minutes).84
d. Sexual and Physical Abuse Questionnaire (SPAQ): A validated questionnaire 
that characterizes the type and age of occurrence of traumatic life experiences 
(5 minutes).85
e.
Beck Depression Inventory –II (BDI-II): A 21-question validated self-report 
inventory for measuring the severity of depression (3-5 minutes).86
f.
Beck Anxiety Inventory (BAI): a 21-question validated self-report inventory 
for measuring the severity of anxiety (3-5 minutes).87
g. Center for Epidemiologic Studies Depression Scale – Revised (CESD-R): A 
20-item validated self-report inventory for screening for depression (3-5 
minutes).
h. Gratitude Questionnaire –Six Item Form: A six-item self-report questionnaire 
designed to assess individual differences in the proneness to experience 
gratitude in daily life. The gratitude questionnaire will only be administered to 
the PI-ME/CFS participants.88
4.5.8 : Neurocognitive testing
a.
Wechsler Test of Adult Reading (WTAR) (The Psychological Corporation, 2001) 
requires the examinee to read words aloud.89
b. Wechsler Adult Intelligence Scale – Fourth Edition (WAIS-IV) subtests including 
Coding, Symbol Search and Digit Span will be administered. For these subtests, 
examinees memorize strings of numbers or complete speeded tasks involving 
unfamiliar symbols.90
c.
Test of Variables of Attention requires examinees to rapidly respond using a 
button press to certain target stimuli and not distractor stimuli.91
d. Hopkins Verbal Learning Test-Revised (HVLT-R) requires examinees to 
remember words that were read to them earlier.92
e.
Brief Visual Memory Test-Revised (BVMT-R) requires examinees to remember 
designs that were shown to them earlier.93
f.
Wisconsin Card Sort Test (WCST-64) requires examinees to utilize corrective 
feedback to learn how to sort cards.94
g. Controlled Oral Word Association Test (COWAT; FAS and Animals) requires 
examinees to generate words to various cues.95

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h. Paced Auditory Serial Addition Test (PASAT) requires examinees to rapidly 
perform serial addition.96
i.
Grooved Pegboard Test requires examinees to rapidly insert pegs in holes.97
j.
Word Memory Test requires the examinee to view words and later remember 
them.98
k. B Test asks the examinee to rapidly discriminate between letter stimuli.99
l.
Dot Counting Test asks the examinee to count dots as rapidly as possible.100
m. Minnesota Multiphasic Personality Inventory – 2 Restructured Form (MPI2-RF) 
requires examinees to complete true-false items that best describes themselves.101
n. Effort-Expenditure for Rewards Task (EEfRT) is a multi-trial game in which 
participants are offered a choice between two task difficulty levels for a reward. It 
is fully described in Section 4.6.19.
The complete battery will take about 2 to 3 hours.
4.5.9 : Occupational Therapy Evaluation (only ME/CFS): Only ME/CFS participants may 
have an evaluation from occupational therapy. A licensed occupational therapist will 
perform an open-ended, focused interview intended to elicit perceptions of fatigue and its 
effect on function, in an unstructured format. The interviewer is free to ask additional 
questions to more fully understand the participant. Interviews to be digitally recorded and 
transcribed.
The Activity Card Sort – 2nd edition will also be administered.102 This is an instrument 
used for assessing participation in occupational performance of instrumental, social- 
cultural, and leisure activities. The tool uses a Q sort method in which respondents sort 
89 cards depicting people engaged in various activities into 5 mutually exclusive 
categories, such as “never done,” “continue to do,” “recently added,” “given up,” and “do 
less.” The Activity Card Sort yields a total score and four domain scores which can be 
used to determine a "retained activity level" score that is the percentage of activities in 
which a person is currently engaged in divided by those with whom he or she was 
involved in the past. The occupational therapy evaluation will take 60-90 minutes.
4.5.10 : Magnetic Resonance Imaging (MRI): A MRI of bilateral thighs and a structural 
brain MRI will be performed. Scanning will be performed either at Clinical Center 
Radiology or on a dedicated MRI scanner in the NMR Center. Scans may include clinical 
anatomic images, MPRAGE, DWI (for DTI) and T2w sequences. For the structural brain 
MRI, intravenous gadolinium contrast to determine cortical enhancement or meningeal 
inflammation will be given if there are no contraindications. MRI scans may include 
intravenous administration of an FDA-approved, macrocyclic, gadolinium-based MRI 
contrast agent (if applicable: including in healthy volunteers). Unless otherwise specified 
in the protocol, contrast agents will be used at FDA-approved doses. NIH Clinical Center 
Radiology and Imaging Sciences guidelines for gadolinium administration will be 
followed. Under certain circumstances, participants in this study may receive a linear 
chelate. Examples of situations in which this may occur include the following: (1) prior 
sensitivity to all macrocyclic agents; (2) participation in a longitudinal study in which a 
linear chelate was previously given, there is a clear advantage to maintaining the same

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gadolinium protocol, and T1 signal change in the basal ganglia or dentate nuclei was not 
observed; or (3) after consultation with, and approval by, a radiologist credentialed at the 
NIH Clinical Center. A list of the instances in which a linear chelate was administered, 
along with the justification for its administration, will be provided to the IRB at the time 
of continuing review. NIH Clinical Center Radiology and Imaging Sciences guidelines 
for gadolinium administration will be followed. Under certain circumstances, participants 
in this study may receive a linear chelate. Examples of situations in which this may occur 
include the following: (1) prior sensitivity to all macrocyclic agents; (2) participation in a 
longitudinal study in which a linear chelate was previously given, there is a clear 
advantage to maintaining the same gadolinium protocol, and T1 signal change in the 
basal ganglia or dentate nuclei was not observed; (3) after consultation with, and approval 
by, a radiologist credentialed at the NIH Clinical Center. A list of the instances in which a 
linear chelate was administered, along with the justification for its administration, will be 
provided to the IRB at the time of continuing review.
All NMR Center scans will be performed with dedicated radiofrequency coils that have 
received approval from the NIH Safety Committee. All women of childbearing potential 
will have a urine pregnancy test performed no more than 24 hours before each MRI scan. 
The blood and/or urine collections will be completed prior to MRI to screen for renal 
dysfunction or pregnancy that would be contraindicated for MRI (45-60 minutes).
4.5.11 : Muscle strength testing: Hand-held dynamometers are simple, portable, and 
relatively inexpensive method to measure isometric muscle strength. It has been shown to 
be a reliable tool as it correlates well with muscle strengths (hip abductors, knee 
extensors, pectorals, shoulder flexors) of individuals with COPD.103 Grip strength will be 
measured on both hands in a neutral position of the arm, forearm, and wrist. Two 
consecutive attempts at 1-minute intervals will be measured in kilograms with the hand- 
held dynamometer. Each attempt will require a participant to exert a maximum possible 
grip force for about 5 seconds. After a minute of rest, the third attempt will measure 
fatigue resistance by asking the participant to hold the maximum hand grip as long as 
able until the 50% maximum grip force (based on the highest maximum hand grip force 
obtained from the first two attempts) is reached, which is often reached in less than a 
minute. The test will be repeated on the other hand. The same dynamometer will be used 
for all participants (30 minutes).
4.5.12 : Activity Monitor and Fatigue Diary Instruction (ME/CFS, HV): Eligible 
participants will receive instruction from a study investigator regarding the use of an 
activity monitor and a fatigue diary at home prior to the participant’s return to the 
Clinical Center for the inpatient admission. The study investigator will explain how and 
when to use the activity monitor and fatigue diary and how it will be mailed to the 
participant at home approximately two weeks prior to the scheduled admission. The 
fatigue diary is designed to occur in conjunction with activity monitoring. Measurements 
will be made at intervals throughout the day, starting at the time of awakening and 
concluding at bedtime. The participant will use the activity monitor for at least five days 
prior to the scheduled admission and throughout the inpatient stay. It will be worn 
continuously (i.e. while awake and while asleep) during this time period. It can be

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removed if the patient showers, bathes, or swims. Medication withdrawal will be 
complete prior to activity monitoring. COVID-19 HV participants will not receive this 
instruction.
4.5.13 : Nutritional Assessment:
Nutrition staff will either meet in person with the participants during their Visit 1 
admission or contact consented participants by telephone prior to the Visit 2 admission. 
During this meeting, participants will be instructed on food record keeping and educated 
about the controlled metabolic menu that they will have for Visit 2. Selections will be 
made for the menus that they prefer to have during Visit 2 and food allergies and 
intolerances will be assessed. Changes to controlled menus will only be made if possible 
to accommodate food allergies. COVID-19 HV participants will not receive this 
assessment.
All participants will fill out an internet-based food frequency questionnaire (Diet History 
Questionnaire (DHQII), National Cancer Institute, Rockville, MD) which asks them 134 
questions regarding their dietary intake over the past year and 8 questions about dietary 
supplement intake. This questionnaire is completed online and typically takes 
approximately 45-60 minutes to complete. This may be completed during Visit 1 or Visit 
2.
Participants will be asked to provide detailed food records for 7 days prior to their 
admission during the same week that they are wearing the activity monitor and recording 
the fatigue diary. These food records will be reviewed by NIH nutrition staff for accuracy 
using 3-dimensional models and then entered into Nutrition Data Systems for Research 
(NDSR, University of Minnesota, Minneapolis, MN) to obtain nutrient intake. COVID-
19 HV participants will not receive this assessment.
4.5.14 : Saliva Sample Collection: Saliva will be collected using 1-2 salivary swabs taken 
from the anterior floor of the mouth and near the Stenson’s ducts. Samples will be 
collected from a clean mouth at least two hours after the participant’s last meal. It takes 
about one minute to collect a sample.
4.5.15 : Buccal Swab Sample Collection: Buccal swabs will be collected using a brush 
rotated against the buccal surface. Buccal swabs will be used for microbiome and 
micronutrient profiling. (<1 minute)
4.5.16 : Stool sample collection: A fresh stool sample will be collected in sterile cup and 
frozen immediately. Stool samples will be used for microbiome and micronutrient 
profiling.
4.5.17 : Holter monitoring: Heart rate variability will be assessed using standardized 
Clinical Center EKG/Holter Monitor data, which will be collected per NIH Cardiology 
standard routines. The EKG monitoring will last less than 24 hours. 
4.5.18 : Lumbar puncture: All participants will undergo an LP to collect cerebrospinal 
fluid (CSF) for research. The LP will be done at the Clinical Center at the bedside or in

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Radiology if done under fluoroscopy. Inpatients will recover on the unit and outpatients 
will recover in the outpatient day hospital. For outpatients, study clinical team member 
will contact the subject the next day to assess for potential adverse events and provide 
contact information if problems arise after the procedure. The NIH Radiation Safety 
Committee guidelines will be followed for participants undergoing LP under fluoroscopy. 
Sedation with low-dose benzodiazepines may be used for subjects with anxiety during the 
LP. Analysis of CSF may include (but will not be limited to): opening pressure, cell 
count, total protein, glucose, PCR for pathogens, cytokine assays, lipid profile, and flow 
cytometry for phenotyping of immune cells (CD4 and CD8 T cells including central 
memory, central effector, regulatory and naïve T cell subsets, B cells and monocytes), 
cytokine/chemokine profile, growth factors, proteome and metabolome, autoantibodies to 
brain antigens and neurotransmitters. CSF will also be aliquoted and stored for future use. 
Additionally, specialized laboratory testing such as detection of autoantibodies to novel 
antigens will be performed as needed. The upper limit of CSF withdrawn will be 24 ml. 
(30-60 minutes)
4.5.19 : Autonomic testing (optional): All participants may undergo provocative 
autonomic testing, which includes:

Breathing maneuvers:
o
Deep breathing: The participant will lie supine with head on pillow and 
breathes deeply at a rate of 5 to 6 breaths per minute for 3 minutes.
o
The Valsalva maneuver: Assessment of beat-to-beat blood pressure and heart 
rate during and after performance of the Valsalva maneuver is a well-accepted 
autonomic function test.1 The participant lies supine on a tilt table with head 
on pillow. The participant then blows or strains against a resistance for 12 
seconds at 30 mmHg and then relaxes. If a “square wave” phenomenon is 
observed, the subject may be tilted at 20 degrees head up and the procedure 
repeated. At least 3 Valsalva maneuvers will be performed. The maneuvers 
will be repeated (at least 1 minute between repetitions) until a technically 
adequate recording is obtained. If the participant cannot perform a technically 
adequate Valsalva maneuver by blowing against a resistance, then a Valsalva 
maneuver may be done alternatively by having the subject strain against a 
closed glottis.

Head-up tilt table testing: The participant will lie supine for at least 15 minutes 
after placement of monitoring leads. Baseline hemodynamic measures are taken 
and blood is drawn. Then the participant is tilted head-up according to the 
following procedures:
o
The participant is tilted head up at a 70-degree angle.
o
At approximately 4-minute intervals, hemodynamic measures are 
taken and blood is drawn.
o
Hemodynamic measures may also be taken and blood drawn if the 
investigative team feels that neurally mediated hypotension or syncope 
may occur soon (e.g., if the participant talks or skin electrical 
conductance increases, or the blood pressure becomes notably more 
variable).

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o
Upright tilt is continued for up to 40 minutes. Tilting will be ceased in 
the event of sudden bradycardia, hypotension, or syncope.
o
After the tilting the participant is brought to the supine position and 
observed for at least 10 minutes.
o
At 10 minutes of recovery, hemodynamic measures are taken and a 
blood sample drawn.

Hemodynamic and non-invasive monitoring: For monitoring, 
electrocardiographic and impedance cardiographic leads may be applied to the 
chest, abdomen, or neck; a laser-Doppler flowmeter placed on a finger; a skin 
thermistor placed on a finger; skin conductance electrodes placed on two fingers; 
a finger cuff applied for non-invasive measurement of beat-to-beat blood 
pressure; an automated blood pressure cuff applied for standard measurement of 
brachial blood pressure; a forearm plethysmograph transducer and rapid-inflation 
brachial cuff placed around the same arm for forearm blood flow measurements; a 
device applied for monitoring respiration; or a pulse oximeter applied to a finger. 
One or more of these devices may be omitted if there are technical or practical 
problems, without the omission being considered a protocol deviation. Of these 
non-invasive monitors, the following are not standard, though they are validated, 
for autonomic testing: forearm plethysmography, impedance cardiography, laser- 
Doppler flowmetry.

Blood sampling: Blood may be sampled through direct venipuncture or 3-way 
stopcocks attached to indwelling i.v. catheters, for assays of catecholamines and 
other neurochemicals. Autonomic disturbance from decreased ganglionic 
neurotransmission, identified by abnormal hemodynamic responses to the 
Valsalva maneuver, low plasma norepinephrine levels, and neuroimaging 
evidence for intact postganglionic sympathetic nerves, can result from a 
circulating antibody to the ganglionic nicotinic acetylcholine receptor. Plasma 
from patients with this constellation of clinical laboratory findings may be 
assayed for such an antibody.

Urine collection: Urine may be collected from participants and will be analyzed to 
understand catecholamine and other neurochemical metabolism. Urine may be 
collected at multiple time points, for example, before and after tilt table testing. 
Participants unable or unwilling to give urine samples will still be able to 
participate in other aspects of the protocol.
Participants will not undergo autonomic testing if they are taking medications that 
would interfere with interpretation of the results, have inner ear injury or disease, are 
known to be unable to tolerate autonomic testing, or are unwilling to participate 
related to cumulative procedural burden.
4.5.20 : Immune Cell collection: All participants will be given a choice to undergo 
cytapheresis or an immune cell blood collection.

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
Cytapheresis (Option A): Participants choosing cytapheresis will undergo 
apheresis to collect of immune cells for immunology testing. Cytapheresis takes 
about 2.5 hours

Immune cell blood collection (Option B): For participants unwilling or unable to 
undergo cytapheresis, a blood draw will be performed instead. Up to 100mL of 
blood will be drawn for the storage of serum, plasma, and PBMCs. Blood draws 
take about 30 minutes. Typically, no more than 150ml of blood will be drawn in 
a single day. No more than 550ml of blood will be drawn over an 8-week period, 
per NIH guidelines.
Participants may be asked to undergo immune cell collection procedures an additional 
time during the period of their study participation.
4.5.21 : Medication Review and Wash-out Planning (ME/CFS only): Medications that 
may interfere with measures of brain excitability have the potential to confound several 
of the proposed study results. Examples of such medications include tricyclic 
antidepressants, hypnotic, antiepileptic, antipsychotic medication, stimulants, 
antihistamines, muscle relaxants, dopaminergic medications, opiates, and sleep 
medications. In clinical practice, participants with PI-ME/CFS often take large numbers 
of medications for their disorders, including those that might impact brain excitability. 
However, there are currently no medications that have been demonstrated to effectively 
treat PI-ME/CFS, and many of the medications these patients take have a negligible 
impact on the symptom of their illness. Therefore, to increase study homogeneity and 
reduce potential confounding from medications, a medication review will be conducted 
and a medication wash-out plan determined for its participants prior to participation in 
the exercise stress visit or the optional fMRI and TMS procedures.
Medication wash-out will be considered for any medication that is not medically 
necessary in the opinion of study investigators and the participant’s physician care 
providers. Examples include medications used only on an ‘as needed’ basis (PRN), 
medicines used solely to address symptoms (i.e. fatigue, pain, sleep quality, mood), 
syndromic diagnoses (i.e. fibromyalgia, ME/CFS), medications prescribed for 
questionable indications, and medications given at sub-therapeutic doses. Participants 
taking stable doses of medications for medical diseases or psychiatric diagnoses will not 
be withdrawn from their medications. Participants taking medications that cannot be 
withdrawn as an outpatient or would place participants at high-risk for withdrawal-related 
complications (i.e. long-acting opiates or benzodiazepines), will not undergo withdrawal 
and will be excluded from study participation.
A study LIP will develop the medication withdrawal plan in conjunction with the 
participant and relevant physicians. All withdrawal plans will require the agreement and 
full cooperation with the participant’s primary care physician. If psychiatric medications 
prescribed by the participant’s psychiatrist are being withdrawn, agreement and full 
cooperation from the psychiatrist will also be obtained. Participant will be informed that

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if they develop urgent or life-threatening problems during the medication withdrawal that 
they should immediately go to an Emergency Room for immediate evaluation and request 
that the evaluating physicians contact the study investigators and physician care 
providers. For non-urgent problems, participants will be instructed to call either the study 
investigators or local physician care providers.
The nature of the medication taper will depend on the particularities of the medication. 
Some medications will be able to be stopped immediately. Other medications may 
require a tapering regimen over days to weeks. The longest taper anticipated will be with 
the SSRI/SNRI class of medications which can require up to 6 weeks to taper. The timing 
of the medication withdrawal will depend on the length of time required to successfully 
withdraw a medication, the number of medications that require withdrawal, and the 
anticipated time for the inpatient visit. In the case of participants taking multiple 
medications, withdrawal will likely be sequential. The goal is for participants to be off of 
potentially interfering medications for at least 5 plasma half-lives prior to their inpatient 
visit.
Participants will be monitored at a minimum of weekly per telephone during outpatient 
withdrawal period by a clinically-credentialed member of the study staff. An AE baseline 
assessment will be performed per phone at the time of beginning of tapering, at least once 
during the tapering period, and at least once during the period off study drug prior to 
admission. Closer monitoring may occur at the initiation of the withdrawal or if the 
participant develops withdrawal issues. Participants with severe withdrawal-related AEs 
will receive appropriate care from their involved physicians, which may include 
hospitalization and/or restarting the medication. Participants unable to withdraw off 
medications will be excluded from the study. After completion of the exercise stress visit, 
participants may be titrated back onto their baseline type and level of medication in 
conjunction with the care of their physician care providers.
4.5.22 : Sedation during LP (optional): Sedation with low-dose benzodiazepines may be 
used for participants with anxiety during the LP. All participants receiving sedation 
during the study will be monitored for side effects.
4.5.23 : Fluoroscopic-guided LP (optional): If the study investigators determine that the 
LP should be done under fluoroscopy because of a participant’s body habitus, the 
procedure will be done in Radiology at the Clinical Center. The fluoroscopic-guided LP 
involves exposure to radiation. The radiation exposure is not required for medical care 
and is for research purposes only.
4.5.24 : Medical, Psychiatric, Sleep Service, and Rehabilitation Consultation (optional): 
Medical, psychiatric, sleep and/or rehabilitation consultation may be requested if it is 
clinically required or desirable in order to provide for the inpatient needs of study 
participants.
4.5.25 : Case adjudication: The determination of PI-ME/CFS status will occur after the 
completion of all clinical and research measurements of the phenotyping visit. Relevant

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descriptive information, medical documentation, and clinical laboratory measurements 
will be de-identified and compiled into an adjudication packet. These adjudication 
packets will then be submitted to the protocol’s adjudication panel for review and case 
status determination.
The adjudication panel will be chaired by the primary investigator (Dr. Avindra Nath). 
Five other members will serve on the panel. Panel members will be current or previously 
licensed independent practitioners with community-recognized experience with the 
diagnosis and care of persons with ME/CFS that have also published on the subject in 
peer-reviewed journals. The names of the Adjudication Board members are listed in 
Appendix 26.10. The primary investigator reserves the right to invite and dismiss panel 
members as best serves the needs of the protocol.
The case adjudication packet for each participant will be sent to the chairman and each 
adjudicator for review. All adjudicators will be asked to review each PI-ME/CFS 
participant’s case packet within 6 weeks of the completion of the phenotyping visit.
Adjudicators will review the packets and vote as to whether the reviewed case is or is not 
consistent with the study criteria for PI-ME/CFS. Adjudicators will be given two weeks 
in which to conduct their review and vote. Votes will be cast using a password-protected 
online voting system. A unanimous decision is one in which all adjudicators responding 
within the 2-week response window all agree that a case meets the protocol’s PI-ME/CFS 
criteria. The chairman and at least two members of the adjudication panel must vote to be 
considered a voting quorum.
In the case of diagnostic disagreement, a teleconference will be arranged to discuss the 
case and determine if consensus can be reached. At least three members of the 
adjudication panel, including the chairman and all dissenting panel members, must be 
part of the teleconference to be considered a voting quorum. In cases where adjudicators 
do not agree, the individual cases will not be considered a case of PI-ME/CFS and 
individual case opinions will be recorded.
To be invited back for the exercise stress visit, a PI-ME/CFS participant must be 
unanimously adjudicated to meet the inclusion/exclusion criteria detailed in Sections 3.2 
and 3.3. In particular, the adjudication committee will review the inclusion and exclusion 
criteria where clinical judgment is required. Examples of such include the determination 
of whether a case is post-infectious, if symptoms are related to an alternative diagnosis or 
related to concomitant medical and/or psychological illness, and if a case meets published 
diagnostic criteria.
Participants that do not meet these criteria will be notified that participation in the study 
has ended. Participants that do not meet criteria for participation in the exercise stress 
visit will be provided one or more of the following general explanations:

Members of the adjudication panel thought that an alternative diagnosis that was 
discovered during phenotyping could be related to ME/CFS symptoms

Members of the adjudication panel thought that other medical issues are 
contributing, at least in part, to ME/CFS symptoms

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
Members of the adjudication panel did not reach consensus agreement that 
ME/CFS symptoms started after an infection

Members of the adjudication panel found that the participant did not meet the 
published ME/CFS diagnostic criteria used in this study

Members of the adjudication panel found that the participant did not meet the 
protocol’s symptom severity criteria

The level of functional impairment observed during the phenotyping visit would 
interfere with being able to perform the exercise stress tasks
The case status of healthy volunteers and COVID-19 HV participants will be 
adjudicated by the study team, not the adjudication panel.
4.6 Study procedures: Exercise Stress visit (Visit 2)
PI-ME/CFS and HV participants deemed eligible for further study participation after the 
phenotyping visit and case adjudication process will be scheduled for an exercise stress 
visit. The exercise stress visit will be an inpatient admission lasting approximately 5-10 
days, with the possibility of extending admissions for clinically-related reasons. The 
home ward for participants during Visit 1 will be the metabolic unit and the hospital bed 
for the admission will be within a metabolic chamber.
Peak exercise testing is the major experimental stimulus for this protocol. It is anticipated 
that performance of this task will induce post-exertional malaise in PI-ME/CFS patients 
but not in either of the control groups.
In this protocol, the period of days prior to the peak exercise test will be referred to as the 
Pre-Exercise period. The day the exercise challenge is administered is referred to as 
Exercise Day 0. Each day following the exercise challenge will be additively referred to 
as a Post-Exercise Day (i.e. 24 hours after peak exercise testing would be Exercise Day 
1; 72 hours after exercise would be Exercise Day 3).
PI-ME/CFS participants are expected to develop post-exertional malaise within 24-48 
hours. All participants will be observed and measured for 72 hours after exercising (Post- 
exercise day 3).
The NIH team will not provide primary treatment for ME/CFS or other medical problems 
that may arise during this study except in the event of medical necessity due to acuity of 
illness or lack of access to timely and appropriate care.
The procedures performed during the exercise stress visit will be scheduled based on 
availability of testing and investigators and will be scheduled to minimize patient 
inconvenience. Optional procedures for research purposes may also be performed during 
the exercise stress visit. The optional procedures will be scheduled based on availability 
of testing and investigators and will be scheduled to minimize patient inconvenience. A 
study schematic is attached as Appendix 24.1.

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Urine pregnancy testing will always be done prior to MRI, fMRI, TMS, and the LP (if 
done under fluoroscopy) for women of childbearing potential.
4.6.1 : Updated History and Physical Examination
A comprehensive clinical assessment usually lasts 1-2 hours and will include 
measurement of vital signs, and a history and physical examination. Inpatients will be 
seen by a LIP daily. Progress notes will be documented in CRIS.
4.6.2 : Intravenous line placement
An intravenous line will be placed and may be kept in place during the hospital 
admission. This will allow for obtaining serial blood samples. Intravenous lines will be 
changed per clinical center protocol. IV placement takes between 5-30 minutes.
4.6.3 : Blood collection
Blood will be collected at various times during the inpatient admission. Blood draws 
take about 30 minutes. A schedule of blood draws is depicted in the study schematic 
(Appendix 24.1). No more than 250 ml of blood will be drawn in a single day. No more 
than 550 ml of blood will be drawn over an 8-week period, per NIH guidelines.
Blood will be serially drawn before and after CPET. This is fully described in Section 
4.6.13.
Blood may optionally be drawn for tests that may include, but will not be limited to:
Acute care panel
PT/PTT (needed prior to LP)
Hepatic panel
Complete blood count with differential
Serum pregnancy testing (for women of childbearing potential if problems arise 
with obtaining urine pregnancy testing).
4.6.4 : Urine collection
A urinalysis for urine toxicology will be completed during the inpatient admission and 
may be used to test for pregnancy in women of childbearing age. A sample of urine will 
also be collected and stored for future use. Collecting a urine sample takes up to 5 
minutes.
4.6.5 : Saliva sample collection
Samples will be collected by participants chewing briefly on collection swabs (or simply 
spitting into a collection vial) at three times points (At 9pm, at 7:30 am, and 12pm).
Samples will be immediately placed on dry ice until they are able to be stored in a -80°C 
freezer. These measures will be made on the same days as MRI and TMS testing during 
the Pre-Exercise period. A third set of salivary cortisol measures will be made on the 
same day as exercise testing. A fourth and fifth set of salivary cortisol measures will be 
made on the same days as MRI and TMS testing during the Post-Exercise period.
Salivary cortisol testing will be completed on these samples according to standardized

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testing by a commercial laboratory. Additionally, aliquots of saliva may be stored for 
future use (1 minute).
4.6.6 : Patient-reported outcome
Patient reported outcome measurements will be made during the exercise stress 
admission. Select questionnaires may be performed at multiple time points in order to 
determine subjective change before and after the peak exercise intervention.
CFS Symptom Inventory: Visual Analogue Scale for CFS Symptoms (VAS-CFS 
Symptoms) consists of 10 items related to physical fatigue, mental fatigue or mental fog, 
muscle aches, join aches, lightheadedness, “flu-like” symptoms, sore throat, 
gastrointestinal discomfort, shortness of breath, and environmental sensitivity. This 
questionnaire will be administered at multiple time points during the inpatient stay (2 
minutes).69
The Brief Pain Inventory (BPI): a validated measurement of pain severity and pain 
interference. Both are scored on 0 -10 point scales with higher scores indicating more 
severe pain. This questionnaire will be administered at multiple time points during the 
inpatient stay (10 minutes).104
The approximate time points at which these tests will be administered include: up to 4 
hours before CPET, up to one hour after CPET, 4-6 hours after CPET, 20-28 hours after 
CPET, 44-52 hours after CPET, and 68-76 hours after CPET.
Other tests to be administered only once include:
1. Multidimensional Fatigue Inventory (MFI): The MFI is a validated 20-item self- 
report instrument designed to measure fatigue severity.70 The items are designed 
to assess general, physical, emotional, and mental manifestations of fatigue as 
well as vigor, an estimate of the patient’s energy level. The MFI-20 has been 
validated in ME/CFS patients (10 minutes).71
2. Patient Reported Outcomes Measurement Information System – Short Forms 
(PROMIS-SF) PROMIS is a system of highly reliable, precise measures of 
patient–reported health status for physical, mental, and social wellbeing. These 
computer adaptive tests are generally less than 6 questions and developed from 
more than 1000 datasets from multiple disease populations including cancer, heart 
disease, rheumatoid and osteoarthritis, psychiatric conditions, spinal cord injury, 
and chronic obstructive pulmonary disease. Initial psychometric properties 
showed internal consistency reliability coefficient of 0.81. PROMIS-SF measures 
that are part of the Core Screening Evaluation include Fatigue, Pain Behavior, 
Pain Interference, Pain Intensity, Global Health, Emotional Distress – Anxiety, 
Emotional Distress- Depression, Sleep-Related Impairment and Sleep 
Disturbances (10 minutes).72
3. The National Institutes of Health – Brief Fatigue Inventory (NIH-BFI) is a 7-item 
clinician-administered questionnaire. To assess reliability, a principle component

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analysis was applied and yielded a single component. Further, the NIH-BFI 
produced Cronbach alphas ranging from .81-.88. The NIH-BFI is strongly 
correlated with fatigue items from validated clinician-administered depression and 
mania scales (2 minutes).73
4. The McGill Pain Questionnaire (MPQ): a list of 20 groups of adjectives to 
describe sensory, affective and evaluative aspects of pain. Using the MPQ, 
participants will be asked to describe their pain experience (2 minutes).74
5. The Neuropathic Pain Scale (NPS): a questionnaire designed to assess distinct 
qualities associated with neuropathic pain. It measures both the quality and the 
intensity of the neuropathic sensations (5 minutes).75
6. Polysymptomatic Distress Scale: Polysymptomatic Distress Scale (PSD): The 
polysymptomatic distress scale is a self-administered instrument that determines 
both the distribution of painful areas across the body and an estimate of related 
symptom burden. Evidence supports the reliability and validity of the PSD in the 
general population (5 minutes).76
7. Patient Health Questionnaire-15 (PHQ-15): A validated questionnaire used to 
assess somatic symptom severity and the potential presence of somatization and 
somatoform disorders (3-5 minutes).77
8. PROMIS sleep and wake disturbances. These short forms are 8-item 
questionnaires developed from more than 1000 datasets from multiple disease 
populations including cancer, heart disease, rheumatoid and osteoarthritis, 
psychiatric conditions, spinal cord injury, and chronic obstructive pulmonary 
disease. Initial psychometric properties showed internal consistency reliability 
coefficient of 0.81 (4 minutes).72
9. Pittsburgh Sleep Quality Index (PSQI): The PSQI measures sleep quality over a 
1-month period with 19 questions in 7 clinically-derived component scores. This 
instrument has a sensitivity of 89.6% and a specificity of 86.5% for identifying a 
sleep disorder (PSQI<5) in a clinical sample (15 minutes).78
10. Fatigue Catastrophizing Scale is a 10-item paper/pencil questionnaire to measure 
catastrophizing related to the fatigue experience. This questionnaire takes less 
than two minutes to complete. It uses a 5-point rating scale (1= never true) to (5
= all the time true) with proven high internal consistency reliability (coefficient 
alpha = 0.85 – 0.92) (2 minutes).79
11. The Multiple Ability Self-Report Questionnaire (MASQ): A 38-item 
questionnaire that assesses the subjective appraisal of cognitive difficulties in five 
cognitive domains: language, visual-perceptual ability, verbal memory, visual- 
spatial memory, and attention/concentration. The MASQ sub-scales are scored on

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an 8–40 point scale with high scores indicating greater perceived difficulties (10 
minutes). 80
12. Short-Form 36 (SF-36): The SF-36 is easily administered and reliably reflects 
health-related quality of life outcomes. This questionnaire has 36-items that assess 
eight health issues: physical functioning, bodily pain, role limitations due to 
physical health problems, role limitations due to personal or emotional problems, 
general mental health, social functioning, energy/fatigue, and general health 
perceptions. The SF-36 has been tested and validated extensively in a number of 
clinical populations and was developed for self-administration by patients (7-10 
minutes).68
13. Belief about Emotions scale: A validated questionnaire designed to measure the 
beliefs of expressing negative thoughts and feelings (3-5 minutes).81
14. Beck Depression Inventory –II (BDI-II): Beck Depression Inventory –II (BDI-II): 
A 21-question validated self-report inventory for measuring the severity of 
depression (3-5 minutes).86
15. Beck Anxiety Inventory (BAI): a 21-question validated self-report inventory for 
measuring the severity of anxiety (3-5 minutes).87
16. Center for Epidemiologic Studies Depression – Revised (CESD-R): A 20-item 
validated self-report inventory for screening for depression (3-5 minutes).105
4.6.7 : Physical activity monitoring
Participant will arrive for the exercise stress visit having monitored physical activity at 
home with an activity monitor for at least five days. They will continue to be monitored 
throughout the exercise stress visit. An activity log form will be provided to the 
participants so they can enter the incidents (date, time, type of activity) when the monitor 
is removed during the exercise stress visit. This activity log will assist in interpreting the 
data captured by the physical activity monitor. Participants will return the monitor at the 
end of the exercise stress visit.
4.6.8 : Metabolic Chamber
The metabolic chamber is a whole-room indirect calorimeter that allows detailed 
assessment of energy and nutrient balance. Measurements are conducted at stable interior 
(room) temperature, humidity and barometric pressure, which are continuously measured. 
Airtight sampling ports and a four-way air-locking food and specimen passage are 
designed to allow blood draws and specimen retrievals with minimal disturbance to the 
chamber environment. Blood pressure and heart rate values are recorded in the morning 
of the study upon entry into the chamber, and immediately after the completion of the 
respiration chamber stay. Outside air is continuously drawn into the chamber, and the 
flow rate of air at the outlet is measured at using a pneumotachograph with a differential 
manometer. A fraction of the extracted air is analyzed at 1-minute intervals for O2 and 
CO2 concentrations with a thermomagnetic O2 analyzer. This allows for a continuous 
assessment of oxygen consumption (V˙O2), carbon dioxide elimination (V˙CO2), and

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calculation of overall energy expenditure (EE). The ratio between V˙CO2 and V˙O2 (the 
respiratory quotient [RQ]) reflects preference for carbohydrate or fat oxidation.
Continuous measurement of RQ at 1-minute resolution as well as a measurement of 24-hr 
urinary nitrogen excretion enables accurate determination of macronutrient oxidation 
rates.
The metabolic chamber will be the participant’s “hospital bed” during the exercise stress 
visit. Participants will generally spend their downtime between study procedures and 
sleep in the metabolic chamber. In this way, metabolic measurements will be made 
throughout the entire exercise stress visit.
4.6.9 : Body Composition Measurement by Dual Energy X-ray 
Absorptiometry
Upon completion of their stays in the metabolic chamber, subjects will undergo a DEXA 
scan (iDXA, GE Healthcare, Madison WI). With this technique, one can determine total 
and regional body fat and lean soft tissue masses, bone mineral content and density.
DEXA produces photons at two different energy levels, 40 and 70 KeV. The photons 
pass through tissues and attenuate at rates related to elemental composition. Bone 
mineral, with highly attenuating calcium and phosphorous, is readily distinguished from 
soft tissues. The different elemental profiles of fat and bone-mineral free lean 
components allows for the analysis of soft tissue fat content, so that bone mineral, fat, 
and bone mineral fat-free lean components may be resolved. The effective radiation dose 
is less than 1 mrem, which is below the guideline of 5000 mrem per year allowed for 
research subjects by the NIH Radiation Safety Committee. All women of childbearing 
potential will have a urine pregnancy test performed no more than 24 hours before the 
DEXA scan. DEXA scan will not be repeated during Visit 2 if it was performed as part 
of an optional procedure during Visit 1 (Section 5.1.9).
4.6.10 : Metabolic Diet and Nutritional Assessment
All participants will consume a controlled metabolic diet for the entire Exercise Stress 
Visit. Energy needs for weight maintenance will be estimated using the Mifflin St. Jeor 
equation with an activity factor of 1.5.106 The diets will be standardized to have 55% of 
energy from carbohydrate, 30% of energy from fat and 15% of energy from protein. All 
food and food ingredients will be weighed to the nearest gram weight in the NIH CC 
Metabolic Kitchen prior to being served to the participant. Though participants should 
consume all foods provided to them, any food not eaten will be weighed back and then, if 
possible, nutrients will be replaced the same day.
Participants whom did not previously complete the Diet History Questionnaire (DHQII) 
will do so. This questionnaire is completed online and typically takes approximately 45- 
60 minutes to complete. Also, a structured dietary interview to assess dietary avoidances 
will be conducted by NIH nutritional staff (see appendix 26.3).
4.6.11 Resting Energy Expenditure Measurements by Ventilated Hood 
While the metabolic hood is the ‘gold standard’ for metabolic measurements, the 
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As a contingency if a participant is found to be unable to tolerate prolonged time in the 
metabolic chamber, we will perform indirect calorimetry using the ventilated hood 
technique. This entails having the participants breathe into a transparent plastic hood 
placed over the head for up to 40 minutes. Ventilated hood measurements will be timed 
to occur in the morning after a 12 hour fast.
Hood measurements will be made in all participants prior to performing CPET to 
establish a baseline measurement. No further ventilated hood measurements will be made 
if the participant is able to tolerate staying in the metabolic chamber. If a participant is 
found to be unable to tolerate the metabolic chamber, ventilated hood measurements will 
be made each morning that the participant would have otherwise been in the metabolic 
chamber.
4.6.12  Peak Exercise Testing
The testing will be completed at the NIH Clinical Center Rehabilitation Department. All 
participants need to refrain from smoking for at least four hours before the 
cardiopulmonary testing.
Cardiopulmonary Exercise Tests (CPET). CPET will be performed using a cycle 
ergometer. A ramp protocol will be used where the work rate would be gradually 
increased until volitional fatigue is reached by the patient. The target endpoint is 
exertional intolerance defined as the subjects expressed desire to stop cycling despite 
strong verbal encouragement from the testing staff. When the test is stopped, the 
ergometer work rate will be decreased to cool down levels and may be stopped 
immediately at the subject’s request. Thus, the subject determines the stopping point and 
is never pushed beyond their expressed ability to continue. Endpoints for stopping the 
tests will be those recommended by the American Heart Association.107
Supplemental, noninvasive cardiac output (Qt) and muscle oxygenation measurements 
will be made during the CPET. Qt will be measured by bioimpedance plethysmography 
(ZCG) and muscle oxygenation measurements will be measured by near infrared 
spectrometry (NIRs). A fingertip blood pressure cuff may also be used to estimate blood 
pressure and Qt. For further determination of maximal oxygenation values of NIRs 
measurements, a thigh occlusion test will be performed prior to the CPX. Following 
seated rest, an occlusion cuff will be rapidly inflated to and held at ~ 250 mmHg, until a 
plateau is observed in the oxygenation signal (not to exceed 8 minutes). Blood may also 
be sampled through an IV during CPET. Subjects will be allowed to recover sufficiently 
after performing the CPX. The total time required for each CPET is approximately 90 
minutes.
CPET has been shown to reliably induce post-exertional malaise in one small study.108 
To ensure that the exercise task is properly reaching maximal work rates and inducing 
post-exercise malaise, the relevant data from the first five PI-ME/CFS and HV 
participants will be reviewed.

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4.6.13 : Qualitative measurement of Post-Exertional Malaise
Qualitative interviews will help illuminate how PI-ME/CFS patients experience PEM and 
how these experiences differ from those of HV and DLI-A subjects. To date, PEM has 
not been well studied using qualitative methods. Qualitative interviewing offers the 
ability to understand the unique experience of ME/CFS patients and provide a deep, more 
nuanced understanding PEM within their social worlds.
Up to 10 brief 10-30 minute semi-structured qualitative interviews will be conducted 
during the exercise stress visit to capture their first-hand experiences with PEM (if any). 
The actual questions used in the qualitative interview will be developed using a focus 
group methodology previously described (Section 4.3). As the peak of PEM has been 
estimated to occur 24-72 hours after exercise in ME/CFS, qualitative interviews will be 
performed up to 4 hours before CPET, up to one hour after CPET, 4-6 hours after CPET,, 
20-28 hours after CPET, 44-52 hours after CPET, and 68-76 hours after CPET in all 
participants. This serial collection will allow participants to describe their experience 
while it is fresh in their minds and to see if the PEM changes over time.
Interviews may be conducted over the telephone as necessary due to professional 
conflicts and weekend interviewing. An interviewer guide (see Appendix 26.4) will 
structure and direct the interviews so that patients are prompted to explain in detail their 
experiences with PEM, but interviewers will have discretion to modify as appropriate. 
Data collected during qualitative interviews will consist of audio recordings, which will 
be sent to a professional service for transcription via a secure website. Data, recordings, 
and transcriptions will be stored in locked cabinets. Transcriptions will NOT contain any 
identifying information and will be labeled with a number only. Persons who decline to 
be audio-taped will not be included in the qualitative portion of the study.
4.6.14 : Blood measurements before and after CPET
Between 25- 80mL of peripheral blood will be serially drawn during the exercise stress 
visit. The timing of these blood draws will be parallel to the times when qualitative data 
about PEM and questionnaire-based measurements of subjective fatigue are collected. 
The approximate time points at which blood measurements will be collected include: 4 
hours before CPET, up to one hour after CPET, 4-6 hours after CPET, 20-28 hours after 
CPET, and 44-52 hours after CPET and 68-76 hours after CPET in all participants.
No more than 250 ml of blood will be drawn in a single day. No more than 550 ml of 
blood will be drawn over an 8-week period, per NIH guidelines.
4.6.15 : Neurocognitive testing
Neurocognitive testing will be conducted before and after CPET. The TOVA, HVLT-R, 
BVMT-R, and the B-Test and/or Dot Test will be measured pre-exercise and may be 
measured up to four times following CPET.
4.6.16 : Holter monitoring
Heart rate variability will be assessed using standardized Clinical Center EKG/Holter 
Monitor data, which will be collected per NIH Cardiology standard routines. The EKG 
monitoring will last less than 24 hours and will be done pre-exercise and post-exercise.

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4.6.17 : fMRI
fMRI will be completed during the exercise stress visit and will occur on two separate 
days, once before and once after the exercise challenge.  The following imaging 
paradigm will be utilized: The subject will lie in the scanner operating a dynamometer to 
measure the force generated by the muscles of the distal upper limb in a first phase of the 
scan, and a button box for answering a cognitive test in the second phase of the scan. A 
structured set of imaging tasks are planned. An optional MRI-compatible 
electroencephalogram cap may be installed on subject’s head before installing the subject 
in the MRI scanner in order to monitor the cortical activity during fMRI scanning and to 
allow the measurement of the cortico-muscular coupling during the muscular and 
cognitive fatiguing exercises. This may add about 30 min to the setup time.
Each scanning session will begin with a localizing image, an anatomical scan, and 
resting-state BOLD acquisition (20 minutes).
A test of cognitive fatigue will be performed within the MRI environment before the 
muscular fatigue test.  In this study, there will be a sequence of two-choice reaction 
times. The correct choice will be based on a simple cognitive operation and the difficulty 
of the operation will not change over the series. Cognitive fatigue will be deemed present 
if the reaction time increases over the sequence. The test will continue for 10 minutes. 
fMRI will be done for 10 minutes, and the task analyzed as an event related design. The 
time between reaction time trials will be jittered, so that the MR signal can be extracted. 
The behavioral data and the MR data will be related parametrically. Another resting 
BOLD acquisition will be performed after the cognitive task. Even if no significant 
learning is expected related to the task, the subjects will get some training on the task 
prior to the MRI study so the novelty and/or any amount of learning will be further 
minimized. Subjective appraisals of cognitive fatigue will be measured at intervals during 
the cognitive fatigue task (20 minutes).
A test of muscular fatigue will be performed afterwards within the MRI environment. 
The last resting BOLD acquisition for the prior test can be considered the baseline for 
this test. We will determine maximum voluntary contraction (MVC) of the forearm 
muscles, expressed in newtons, from the best of three brief presses on the dynamometer. 
After a resting period, the subject will perform repeated 30-second blocks of isometric 
muscle contractions at half MVC until fatigued, which is defined as the inability to 
maintain at least 40% MVC force for more than 3sec. The subject and the investigators 
will have visual feedback from a computer to monitor force generation. The approximate 
interval between successive exercise periods will be 30 seconds. The period after the last 
exercise will be the recovery period, during which resting BOLD will be acquired.
Acquisition time will be the same length for all subjects, with fatigued subjects simply 
stopping doing the task for the block. Subjective appraisals of muscular fatigue will be 
measured at intervals during the muscular fatigue task (30 minutes).
If MPRAGE, DWI, and T2w images have not been previously collected on a study 
dedicated MRI machine at the NMR Center, they will be obtained at this time. If an MRI-

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compatible cap was installed and further DWI scans are necessary, the cap will be 
removed prior to the DWI scan. (25 minutes).
The fMRI procedures will all be completed on the same scanner at the NMR center and 
should take approximately 90-120 minutes to complete. All women of childbearing 
potential will have a urine pregnancy test performed no more than 24 hours before each 
fMRI session.
4.6.18 : Effort-Expenditure for Rewards Task (EEfRT)
The Effort-Expenditure for Rewards Task109 is a multi-trial game in which participants 
are offered a choice between two task difficulty levels for a reward. The task begins with 
a 1 second fixation cross, followed by a 5s choice period in which the participant is 
informed of the probability of “winning” if the task is completed successfully. After the 
participant chooses the task, a one second “ready” screen is displayed. Next, the 
participant either completes thirty button presses in seven seconds with the dominant 
index finger if they have chosen the easy task, or one-hundred button presses in 21 
seconds using the non-dominant little finger if they have chosen the hard task. Next, the 
participant receives feedback on whether or not they completed the task successfully.
Finally, the participant learns if they have “won”, based upon the probability of winning 
and the successful completion of the task. This process repeats in its entirety for twenty 
minutes. This task will evaluate the theory of value-based decision making in the 
pathophysiology of fatigue by looking for associations between MRI brain measurements 
implicated in reward and measures of fatigue and motivation in our subjects. In the initial 
validation study, trait anhedonia as measured by well-validated self-report measures 
significantly predicted an overall reduced likelihood of making a hard-task choice (b = -
.015, p<0.005).110
Participants are told at the beginning of their task that they will win the dollar amount 
they receive from two of their trials, chosen at random from the computer program (range 
of total winnings is $2.00-$8.60).

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Figure 1. Schematic diagram of a single trial of the modified Effort Expenditure for 
Rewards Task (‘EEfRT’). A. Fixation cross for participant to view, B.  5s choice period 
in which subjects are presented with information regarding the reward magnitude of the 
hard task for that trial and probability of receiving any reward for that trial, C. 1s “ready” 
screen, D. Subjects make rapid button presses to complete the chosen task for 7s (easy 
task) or 21s (hard task), E.  Subjects receive feedback on whether they have completed 
the task, F.  Subjects receive reward feedback as to whether they received a score 
increase for that trial (adapted from Treadway, 2009).
4.6.19 : Lumbar puncture
All participants will undergo an LP to collect cerebrospinal fluid (CSF) for research. The 
LP will be done at the Clinical Center at the bedside or in Radiology if done under 
fluoroscopy. The NIH Radiation Safety Committee guidelines will be followed for 
participants undergoing LP under fluoroscopy.
Analysis of CSF may include (but will 
not be limited to): opening pressure, cell count, total protein, glucose, PCR for 
pathogens, cytokine assays, lipid profile, and flow cytometry for phenotyping of immune 
cells (CD4 and CD8 T cells including central memory, central effector, regulatory and 
naïve T cell subsets, B cells and monocytes), cytokine/chemokine profile, growth factors, 
proteome and metabolome, autoantibodies to brain antigens and neurotransmitters. CSF 
will also be aliquoted and stored for future use. Additionally, specialized laboratory 
testing such as detection of autoantibodies to novel antigens will be performed as needed. 
The upper limit of CSF withdrawn will be 24 ml. LP will be performed during post- 
exercise day 3. (30-60 minutes)
4.6.20 : Stool Sample Collection
All stool produced during the 12 hours prior to CPET and for 72 hours after CPET will 
be collected fresh in a cup and placed in an ice bath or refrigerator immediately. These

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samples will be retrieved, processed, and frozen within 24 hours. Stool samples will be 
used for microbiome and micronutrient profiling.
4.6.21 : Sleep Electrocephalogram
Sleep architecture will be measured before and up to three times after the exercise stress 
test using a portable EEG unit. The portable EEG unit is compatible with use in the 
metabolic chamber. The EEG cap will be placed on the scalp by qualified personnel.
Collodion or gel may be used in conjunction with the cap. The collected data will be used 
for sleep staging and determining within participant and between group alterations in 
sleep macro- and microarchitecture.
5
Descriptions of Optional Produres
5.1.1 Sedation during LP
Sedation with low-dose benzodiazepines may be used for participants with anxiety during 
the LP. All participants receiving sedation during the study will be monitored for side 
effects.
5.1.2 Fluoroscopic-guided LP
If the study investigators determine that the LP should be done under fluoroscopy 
because of a participant’s body habitus, the procedure will be done in Radiology at the 
Clinical Center. The fluoroscopic-guided LP involves exposure to radiation. The 
radiation exposure is not required for medical care and is for research purposes only.
5.1.3 Medical, Psychiatric, and Rehabilitation Consultation
Medical, psychiatric, and/or rehabilitation consultation may be requested if it is clinically 
required or desirable in order to provide for the inpatient needs of study participants.
5.1.4 Whole genome sequencing
All participants will be offered the option of consenting to whole genome sequencing. 
Sequencing will be performed on stored blood samples. A separate informed consent 
form will be used for whole genome sequencing. Samples may or may not be tested, even 
if informed consent for testing is provided.
5.1.5 Skin Biopsies
Participants may be offered the option of undergoing a two-site skin biopsy procedure. 
One skin biopsy will be taken from the distal lateral leg, 10cm proximal to the superior 
margin of the lateral malleolus along the midaxillary line. The second skin biopsy will be 
taken from the distal thigh, 10 cm from the superior margin of the patella. Either the left 
or right leg may be selected for each biopsy. Aseptic technique is to be utilized 
throughout the sampling procedure.
The designated area will be prepared with alcohol wipes or another suitable antiseptic. 
Local anesthetic (i.e. 2% lidocaine with epinephrine) will be infiltrated into the area of 
planned biopsy. Once adequate anesthesia is obtained, a sterile disposable 3mm skin 
punch will be used to obtain a punch skin sample approximately 6mm deep. Using sterile

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disposable forceps, the dermis will be gently lifted and the sample freed using sterile 
disposable scissors. The biopsy sample will then immediately be placed in a sample vial 
containing a fixative agent.
No more than two passes will be attempted at each site. Gentle pressure with sterile gauze 
will be used to address any bleeding. Persistent bleeding (seen in <1:1000 biopsies) may 
be addressed with other hemostatic measures, such as placing gelfoam into the biopsy 
site. Once bleeding stops, the area will be bandaged with sterile bandages.
Only a single two-site skin biopsy will be performed as an optional part of the protocol. 
The skin biopsy may be performed during Visit 1, during Visit 2, or as an additional 
outpatient visit.
5.1.6 Muscle Biopsy
All participants may be offered the option of undergoing a muscle biopsy. The vastus 
lateralis is the site of choice for muscle biopsy. Aseptic technique is to be utilized 
throughout the sampling procedure.
The designated area will be prepared with Betadine, chlorhexidine, or another suitable 
antiseptic. Local anesthetic (i.e. 2% lidocaine without epinephrine) will be infiltrated into 
the area of planned incision with a short #25 needle. Once adequate anesthesia is 
obtained, an incision approximately 0.5 cm long and 2 cm deep will be created. Deeper 
tissue layers can be infiltrated with the local anesthetic utilizing a #20-22 needle.
Orientation of biopsy sample will be in a cross-sectional fashion.
A 5 mm or 6 mm Bergstrom biopsy needle will be used. The procedure goal is to obtain 
at least 200 mg of wet tissue. The number of biopsy 'passes' depends on the sample size. 
No more than four passes will be attempted. An ice pack and pressure will be applied to 
the biopsy site for at least 15 minutes. The incisions will be closed with steri-strips and a 
sterile pressure bandage will be applied. If hemostasis remains an issue after the 
procedure is completed, ice may be applied or the area may be infiltrated with 2% 
lidocaine with epinephrine.
Only a single muscle biopsy will be performed as an optional part of the protocol. The 
muscle biopsy may be performed during Visit 1, during Visit 2, or as an additional 
outpatient visit.
5.1.7 Functional Magnetic Resonance Imaging (fMRI)
Functional MRI may be offered as an optional procedure during Visit 1 or as an 
additional outpatient visit. The fMRI procedure is identical to that being performed 
during Visit 2 and is detailed in Section 4.6.17: functional MRI but will be only be 
performed a single time (rather than before and after an exercise stress). The optional 
fMRI procedure will only be performed after a medication washout (if required) as 
detailed in Section 4.2.21.

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5.1.8 Transcranial Magnetic Stimulation (TMS)
TMS may be offered as an optional procedure for all participants. It may be performed a 
maximum on one time, either during Visit 1, prior to CPET during Visit 2, or as an 
additional outpatient visit. The optional TMS procedure will be performed only after a 
medication washout (if required) as detailed in Section 4.2.21. All women of childbearing 
potential will have a urine pregnancy test performed no more than 24 hours before each 
TMS session. The following paradigm will be utilized:
The subject will sit comfortably on a chair, with the right forearm placed in a rigid-frame 
dynamometer to measure the force generated by the muscles of the distal upper limb. The 
output of the dynamometer will be fed to a computer. We will determine MVC as the 
best of three wrist extensions. We will record the EMG by placing surface electrodes on 
the right forearm. A TMS-compatible EEG cap may be installed, in order to monitor the 
cortical activity during the TMS recordings and to allow the measurement of the cortico- 
muscular coupling during the muscular fatigue exercise.
Using a magnetic stimulator and figure-8 coil, we will determine the optimal position for 
evoking motor potentials (MEP) by holding the coil tangential to the scalp and slightly 
displacing it until the highest MEP amplitude will be recorded from the target muscle.
The positions of subject’s head and TMS coil will be tracked with a neuronavigation 
system, in order to maintain the stimulation position over the hotspot. We will then 
record the TMS Input-Output curve. For all subsequent TMS experiments, we will use 
the S50 stimulus level for the reference MEPs. Also, in this preparatory phase, we will 
record the maximum M-wave by applying electrical stimulation with surface electrodes 
on the nerve innervating the target muscle. All MEP amplitudes will be normalized to 
each subject’s Mmax amplitude.
The TMS evaluation will begin with a block of 100 single pulses delivered every 5 
seconds in order to allow the subsequent analysis of the EEG responses. This battery will 
be followed by a short set of double-pulses for measuring short intracortical inhibition 
(SICI) and intracortical facilitation (ICF).
Then, the subject will perform repeated 30-second periods of isometric muscle 
contractions at half the MVC until fatigued, which is defined as the inability to maintain 
at least 40% MVC force for more than 3 seconds. A short train of single-pulse MEPs 
(elicited every 5 seconds) will be recorded immediately after each 30-second exercise 
period, at 30 seconds after the last MEP recorded in the final exercise period, and at fixed 
intervals thereafter for up to 30 minutes. These single-pulse trains will be alternated with 
blocks of SICI, ICF, and Mmax, that will monitor the effects of muscular fatigue on these 
parameters. The subject and the investigators will have visual feedback from the 
computer to monitor force generation and audio feedback from the electromyograph to 
monitor muscle relaxation and contraction. The period after the last exercise will be the 
recovery period. Because post-exercise MEP facilitation can last a few minutes after the 
last exercise set, post-exercise MEP depression will be defined as the mean of the lowest 
MEP amplitudes recorded either 30 seconds, 2 minutes, or 4 minutes into the subject’s 
recovery period. (2 – 3 hours)

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5.1.9 Metabolic Chamber wearing accelerometers
Spending one night in the metabolic chamber will be offered as an optional procedure 
during Visit 1 or during an additional inpatient visit. Activity monitors (Section 4.6.7) 
will be placed on participants prior to being placed in the metabolic chamber. The 
metabolic chamber procedure is identical to that detailed in Section 4.6.8: Metabolic 
Chamber. As part of this optional procedure, participants will have a DEXA scan 
(Section 4.6.9).
6
End of participation
Participants will complete the study after discharge from the NIH Clinical Center. If 
eligible, individuals enrolled in the study may be offered the possibility of enrolling in 
other NIH protocols as appropriate; however, this is not a requirement for participation in 
this protocol. Participants will be asked if they would be willing to be re-contacted by the 
study investigators for new study opportunities, including additional optional procedures 
added to this protocol in approved Amendments or as separate, related Protocols.
Participants will continue care with the primary care provider throughout the study and 
after end of study participation.
Results of the study procedures available in the participant’s electronic medical record 
will be shared with the patient and with the subject’s physician in a timely fashion via 
written communication in the form of a letter. Should there be a clinical need to 
communicate urgently with the patient or his/her physician, this will be pursued via 
telephone communication in addition to the written communication.
Participants will receive a thank you letter after completing their participation in any of 
the aspect of the research study. These letters are detailed in Appendix 26.8.
7
Management of Data and Samples
7.1 Storage
All collected samples including serum, plasma, PBMCs, CSF, and saliva will be coded 
and stored in secured freezers on the NIH campus. All patients enrolled in this protocol 
will be assigned a sequential code and all biological samples collected for this patient will 
be labeled with the patient’s code, type of the sample, volume, number of cells (if 
indicated) and date of freezing. All study data will be kept on password-protected 
computers on the NIH campus. Only study investigators will have access to the stored 
data and samples. The study investigators will have access to the code key.
Any loss or destruction of samples will be reported to the IRB.
7.2 Data (including genomic data) and sample sharing plan
Samples and data including genomic data may be shared with other NIH protocols, other 
investigators, or databases/repositories, under the following guidelines:

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Data and samples may also be shared with collaborating laboratories at NIH or outside of 
NIH and/or submitted to NIH-designated repositories and databases.
Samples and data will be stripped of identifiers and may be coded (“de-identified”) or 
unlinked from an identifying code (“anonymized”). When coded data is shared, the key 
to the code will not be provided to collaborators but will remain at NIH. Data and 
samples may be shared with investigators and institutions with an FWA or operating 
under the Declaration of Helsinki (DoH) and reported at the time of continuing review. 
Sharing with investigators without an FWA or not operating under the DoH will be 
submitted for prospective IRB approval. Submissions to NIH-sponsored or supported 
databases and repositories will be reported at the time of Continuing Review.
Submission to non-NIH sponsored or supported databases and repositories will be 
submitted for prospective IRB approval.
Required approvals from the collaborating institution will be obtained and materials will 
be shipped in accordance with NIH and federal regulations.
As no large-scale genomic data will be acquired during the course of this protocol, this 
protocol is not subject to the NIH Genomic Data Sharing (GDS) policy.
Under the Human Data Sharing (HDS) policy, in addition to the procedures for sharing 
genomic data, as stated above, de-identified non-genomic study data will be shared with 
the NINDS data repository (data.ninds.nih.gov). Furthermore, applicable (e.g., generated 
via CRIS) identified data will be shared via BTRIS following standard Clinical Center 
operating procedures including BTRIS data access policies. Data to be shared include 
baseline characteristics as well as key study outcome variables in a tabulated format, after 
adequate data cleaning, processing, and quality control. Data sharing will be performed at 
or prior to the time of publication.
8
Additional Considerations
8.1 Research with investigational drugs or devices
MRI evaluations of brain may be performed at field strengths of 1.5T, 3T, and/or 7T, 
under conditions designated by the FDA as constituting nonsignificant risk. MRI scans 
are performed on FDA-approved scanners with approved radiofrequency coils, and their 
use conforms to the corresponding FDA labels. These studies may involve software 
modifications as allowed under 21 CFR 812.2(b)(4), which stipulates that any 
modification “is not for the purpose of determining safety or effectiveness and does not 
put subjects at risk.”
Conventional TMS studies in this protocol are considered non-significant risk (NSR) 
devices and will only be used within published guidelines.21CFR812.2 SR states that a 
significant device
1. is intended as an implant and presents a potential for serious risk to the health, safety, 
or welfare of a subject

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TMS is not an implantable device.
2. is purported or represented to be for a use in supporting or sustaining human life and 
presents a potential for serious risk to the health, safety, or welfare of a subject
TMS is not for use in supporting or sustaining human life. It does not present a potential 
for serious risk to the health, safety, or welfare of participants when used as described in 
this protocol.
3. is for a use of substantial importance in diagnosing, curing, mitigating, or treating 
disease, or otherwise preventing impairment of human health and presents a potential for 
serious risk to the health, safety, or welfare of a subject
TMS, as used under this protocol is not of substantial importance in diagnosing, curing, 
mitigating, or treating disease, or otherwise preventing impairment of human health and 
does not present a potential for serious risk to the health, safety or welfare of a subject.
4. otherwise presents a potential for serious risk to the health, safety or welfare of a 
subject
TMS (single and pair pulses) has been in use for over three decades and have been 
cleared by the FDA for treatment of several disorders. Safety guidelines have been 
developed and updated allowing its dissemination to a wide range of clinical and non- 
clinical settings.111,112 For example, brief, self-limited, seizures were seen in early studies, 
before limits were established for combinations of delivery parameters. However, this 
risk has been reduced to the order of one in every 40,000 sessions, including for 
paradigms like theta-burst stimulation not included in the guidelines.113 In the past 20 
years, the FDA has generally waived pre-IDE inquiries for TMS/rTMS studies on an 
NSR device basis. Hence, the CNS IRB, like most US IRBs, has accepted NSR 
designation for TMS studies within these limitations.
8.2 Gene therapy
Gene therapy will not be used in this study.
9
Risks and Discomforts
The potential risks and discomforts of the study include the following:
9.1 Risks of history and physical examination
There is minimal medical risk or discomfort during the history and physical examination.
9.2 Risks of IV insertion
The risks of an IV include bleeding, infection, or inflammation of the skin and vein with 
pain and swelling. Some people may feel lightheaded.
9.3 Risks of blood collection
Participants may have some discomfort and bruising at the site of needle or IV entry. 
There is a very small risk of fainting. Infection in the area of the needle insertion is rare.

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9.4 Risks of HIV testing
All subjects who test positive for HIV during this study and did not know they were 
positive will be informed of the result. There is a risk of distress for the subject and/or the 
subject’s family from this. Subjects who test positive will be provided additional 
information regarding the diagnosis and management of HIV.
9.5 Risks of urine collection
There is minimal risk or discomfort associated with giving a urine sample. Participants 
who have a positive drug test will be told promptly and will be excluded from the study. 
The results of the drug testing will be noted in the NIH medical record. Participants who 
do not want this information in their medical record should not participate in this study. 
Medical records will only be released with the participant’s written agreement. However, 
insurance companies may require participants to release these records and may not give 
participants insurance if they refuse.
9.6 Risks of symptom assessment/patient reported outcome measures
There is minimal medical risk or discomfort from providing information about ME/CFS 
symptoms and in completing the questionnaires. Some of the questions may make 
participants feel uncomfortable or anxious. Participants may refuse to answer any 
question or to stop a test at any time and for any reason.
9.7 Risks of psychological assessments and psychological inventories
The nature of the questions may be sensitive to some of the participants, in particular 
questions about sexual and personal trauma and suicidality. Participants may choose not 
to answer any question that makes them uncomfortable. If required or requested, 
appropriate referrals will be made as recommended by the psychologists and psychiatrists 
involved with this protocol.
During this study, any psychiatric diagnoses determined as part of this study will be 
discussed along with any recommendations with the participant by a member of the study 
team. A member of the study team may contact the participant’s primary care physician 
to obtain or provide information in some cases.
Participants whose responses to tests and questionnaires suggest non-emergent 
psychiatric or medical issues may be referred to a provider in the community for 
appropriate psychiatric or medical care, unless they are already receiving such care. 
Emergent psychiatric evaluation and treatment will be performed under the guidance of 
personnel from the National Institutes of Mental Health (NIMH) Psychiatry Consultation 
Service. Emergent medical evaluation and treatment will be managed by the NIH Rapid 
Response Team or the NIH Code Blue Team.
If a participant discloses information during completion of the assessment form that 
suggests suicidal or homicidal ideation or intent (i.e., that he or she may be a danger to 
themselves or others) or otherwise suggests a need for psychiatric intervention, the PI of 
the protocol will be notified. If the participant is in imminent danger of harming 
himself/herself or someone else, arrangements will be made for hospitalization outside of 
NIH. If the participant insists upon leaving NIH, we may need to arrange an involuntary

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hospital admission to another facility. If outside hospitalization is necessary, this will 
occur at the participant’s expense.
In other situations where the participant expresses psychiatric or psychological distress 
that is not suicidal or homicidal in nature, the PI or another licensed member of the 
research staff can provide the participant with a referral for psychiatric or other 
evaluation with a clinician and/or for psychiatric assistance with a provider in their home 
community. Moreover, should the questionnaires in this study raise issues a participant 
would like to discuss further; the PI will be available to him or her.
9.8 Risks of neurocognitive testing
The neuropsychological tests may be frustrating or stressful. Participants may refuse to 
answer any question or to stop a test at any time and for any reason. The nature of the 
questions may be sensitive to some of the participants, in particular questions about 
sexual and personal trauma and suicidality.
Participants whose responses to tests and questionnaires suggest non-emergent 
psychiatric or medical issues may be referred to a provider in the community for 
appropriate psychiatric or medical care, unless they are already receiving such care. 
Emergent psychiatric evaluation and treatment will be performed under the guidance of 
personnel from the National Institutes of Mental Health (NIMH) Psychiatry Consultation 
Service. Emergent medical evaluation and treatment will be managed by the NIH Rapid 
Response Team or the NIH Code Blue Team.
If a participant discloses information during completion of the assessment form that 
suggests suicidal or homicidal ideation or intent (i.e., that he or she may be a danger to 
themselves or others) or otherwise suggests a need for psychiatric intervention, the PI of 
the protocol will be notified. If the participant is in imminent danger of harming 
himself/herself or someone else, arrangements will be made for hospitalization outside of 
NIH. If the participant insists upon leaving NIH, we may need to arrange an involuntary 
hospital admission to another facility. If outside hospitalization is necessary, this will 
occur at the participant’s expense.
In other situations where the participant expresses psychiatric or psychological 
distress that is not suicidal or homicidal in nature, the PI or another licensed member 
of the research staff can provide the participant with a referral for psychiatric or other 
evaluation with a clinician and/or for psychiatric assistance with a provider in their 
home community. Moreover, should the questionnaires in this study raise issues a 
participant would like to discuss further; the PI will be available to him or her.
9.9 Risks of Occupational therapy evaluation
There is minimal medical risk or discomfort related to the occupational therapy 
evaluation. Some of the questions may make participants feel uncomfortable or anxious. 
Participants may refuse to answer any question or to stop a test at any time and for any 
reason.

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9.10
Risks of MRI
Participants may be at risk for injury from the MRI magnet if they have some kinds of 
metal in their body. It may be unsafe for participants to have an MRI scan if they have a 
pacemaker or other implanted electrical device, brain stimulator; some types of dental 
implants, aneurysm clips, and metallic prostheses (including metal pins and rods, heart 
valves, and cochlear implants); permanent eyeliner; implanted delivery pump; or shrapnel 
fragments. Welders and metal workers may have small metal fragments in the eye.
Participants will be screened for such metal before having any scan. If they have any, you 
will not receive an MRI scan. If they have a question about metal in their body, they 
should inform the study investigators. Participants will be asked to complete an MRI 
screening form before each MRI scan they have.
All magnetic objects must be removed before entering the MRI scan room. This includes 
items like watches, coins, jewelry, and credit cards.
It is not known if MRI is completely safe for a developing fetus. Therefore, all women 
who are able to get pregnant will have a pregnancy test done no more than 24 hours 
before each MRI scan. The scan will not be done if the pregnancy test is positive.
People with fear of confined spaces may become anxious during an MRI. Those with 
back problems may have back pain or discomfort from lying in the scanner. The noise 
from the scanner is loud enough to damage hearing, especially in people who already 
have hearing loss. Everyone having a research MRI scan will be fitted with hearing 
protection. If the hearing protection comes loose during the scan, participants should let 
us know right away.
Symptoms from the contrast infusion are usually mild and may include feeling hot, 
burning, or
coldness in the arm during the injection, a metallic taste, headache, allergic reactions and 
nausea. In an extremely small number of individuals, more severe symptoms have been 
reported including shortness of breath, wheezing, hives, and lowering of blood pressure. 
Unless specifically allowed by the protocol, participants will not receive gadolinium- 
based contrast agents for research purposes if they have previously had an allergic 
reaction to them. Individuals with a history of anaphylaxis to other agents or chronic 
asthma requiring treatment will not receive gadolinium under this protocol unless they 
have previously received gadolinium and tolerated it well. Participants will be asked 
about such allergic reactions and history of asthma before a contrast agent is 
administered.
People with kidney disease are at risk for a serious reaction to gadolinium contrast called 
“nephrogenic systemic fibrosis,” which has resulted in a very small number of deaths. If 
subjects are 60 years old or greater or have diabetes, kidney disease or liver disease, 
blood work to assess kidney function will be performed within 4 weeks before any MRI 
scan with gadolinium contrast. Participants may not receive gadolinium for a research 
MRI scan if kidney function is not normal. There is no evidence for the potential of 
gadolinium-related toxicity in people with normal kidney function. This protocol follows

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NIH Clinical Center guidelines for kidney-function screening related to gadolinium 
administration.
Most of the gadolinium contrast is eliminated in the urine. However, recent studies have 
found very small amounts of residual gadolinium in the body, including the brain, by 
imaging and at autopsy. Macrocyclic gadolinium-containing contrast agents are 
substantially less likely to leave gadolinium behind than linear agents. The use of 
macrocyclic vs. linear agents in this study is delineated in the procedures section above. 
There is presently no evidence that the retained gadolinium is associated with any 
adverse effects.
9.11
Risks of muscle strength testing
There is minimal medical risk or discomfort for the muscle strength testing.
9.12
Risks of activity monitoring and fatigue diary
There is minimal medical risk or discomfort from the physical activity monitoring and 
recording fatigue levels in a diary.
9.13
Risks of nutritional assessment and metabolic diet
There is minimal medical risk or discomfort from the nutritional assessments and 
maintaining a metabolic diet.
9.14
Risks of saliva collection
There is minimal medical risk or discomfort for providing saliva samples.
9.15
Risks of buccal swab collection
There is minimal medical risk or discomfort for providing buccal swabs.
9.16
Risks of stool sample collection
There is minimal medical risk or discomfort from stool sample collection.
9.17
Risks of Holter monitoring
There is minimal medical risk or discomfort from the Holter monitoring.
9.18
Risks of LP
Participants may have a brief pain or tingling sensation in their legs during the lumbar 
puncture if the needle brushes against a nerve. Participants will be instructed to let the 
study team member know immediately and the needle will be adjusted. Some people get 
a mild backache at the site of needle insertion. About one-third of people have a 
headache for a few days after a lumbar puncture. Usually the headache is not severe and 
improves without treatment other than a mild pain reliever. Headaches lasting longer than 
7 days develop with one in 50 to 200 lumbar punctures and usually improve gradually 
over 2 weeks. In rare cases, headaches persist longer. Prolonged headaches may be due to 
persistent leakage of CSF from the area of the lumbar puncture. If the headache is 
prolonged, participants may get a blood patch. Collecting ~20 ml of CSF per procedure 
represents negligible risk (Evans, et al. 2000). In adults the rate of CSF synthesis is 
approximately 21.5 ml/hour, or approximately 500 ml/24 hours, which represents roughly 
4 times the total volume of CSF in adult patients. Therefore, the maximum volume of 24

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ml of CSF that would be collected will be replenished in its entirety within approximately 
1-1.5 hours after collection.
9.19
Risks of sedation for LP
Sedation with low-dose benzodiazepines is associated with a small risk of respiratory 
depression and an even smaller risk of cardiac arrhythmias. More common risks are: 
sleepiness, dizziness, weakness, unsteadiness, hallucinations, confusion, and rarely an 
allergic reaction causing rash, hives, or swelling.
9.20
Risks of fluoroscopic-guided LP
Fluoroscopy may be used during LP to facilitate placement of the needle into the 
subarachnoid space. Participants who undergo fluoroscopy will be exposed to radiation in 
the amount of 23 mrem, (or 46 mrem for two procedures) which is within the safety limit 
of 5 rem (5000 mrem) per year set by the NIH Radiation Safety Committee for adult 
research subjects. If participants would like more information about radiation and 
examples of exposure levels from other sources, they will be provided with the flyer, “An 
Introduction to Radiation for NIH Research Subjects.”
9.21
Risks of autonomic testing
Dynamic heart rate and blood pressure monitoring during respiratory, positional and 
valsalva maneuvers: The blood pressure monitoring consists of blood pressure sensor 
placed on the finger, this has a squeezing sensation which can be slightly uncomfortable. 
The heart rate monitor, similar to a standard EKG recording, will have three surface leads 
placed on the chest and torso area to noninvasively monitor heart rate. The chest 
expansion monitor will consist of a strap wrapped around the low chest to record the 
breathing pattern. It can be cumbersome but otherwise is painless. The testing maneuvers 
of deep breathing, Valsalva or tilt test may cause lightheadedness, syncope, and 
arrhythmias in some people. Though rhythm disturbances have been noted, the most 
common being extra systoles, the dysrhythmias appear to resolve without medical 
intervention. The Valsalva maneuver can also cause an increase in intraocular and 
intracranial pressure but there has not been any reported complications related to 
intraocular hemorrhage or lens displacement.
9.22
Risks of cytapheresis
Cytapheresis can cause lightheadedness or fainting. Some people have tingling in the 
fingers or around the mouth. The citrate blood thinner used to prevent blood clots during 
apheresis can cause mild muscle cramps. We will slow down the blood flow and/or 
administer calcium carbonate (Tums or similar oral formulation) if participants complain 
of symptoms. If the symptoms do not go away, we will stop the cytapheresis.
9.23
Risks of immune cell blood draw
Participants may have some discomfort and bruising at the site of needle entry. There is a 
very small risk of fainting. Infection in the area of the needle insertion is rare.
9.24
Risks of medication washout
There is a more than minimal risk for the planned tapering off of medications that 
potentially interfere with brain excitability. Participants may experience an increase in

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their typical symptoms or experience symptoms related to the cessation of medications. 
Patients may decide to discontinue with the medication taper if it is too difficult for them 
to tolerate.
9.25
Risks of metabolic chamber
Besides inconveniences that can reasonably be expected as a result of spending an 
extensive time (overnight) in the live-in room calorimeter, there is no risk to subjects’ 
physical health. Claustrophobia is an exclusionary criterion. All subjects will be given an 
opportunity to experience the metabolic chamber prior to enrollment in the study. Risks 
of dual energy x-ray absorptiometry
9.26
Risks of DEXA
For each scan (around 12 minutes), the amount of radiation (effective dose) during the 
DEXA scan is less than one mrem to the whole body. This radiation exposure is below 
the guideline of 5000 mrem per year allowed for research subjects by the NIH Radiation 
Safety Committee The use of the DEXA scan apparatus may cause some minimal 
discomfort in claustrophobic subjects and may cause some minimal back pain in a small 
minority of the individuals.
9.27
Risks of resting energy expenditure measurements by ventilated hood 
The use of the clear plastic ventilation hood may cause some minimal discomfort in 
claustrophobic subjects
9.28
Risks of peak exercise testing
Exercise testing may be risky for participants with cardiac disease so participants with 
known cardiac disease will be excluded from the study. Participants who do not have 
known cardiac disease are believed to be at low risk from exercise tests using a bike or a 
treadmill. The major risk of this type of exercise in general is that someone may have 
unknown or asymptomatic cardiac disease. Even though participants will be examined 
before the study to try to detect the presence of cardiac disease, it is possible to have 
unknown cardiac disease that can lead to angina, arrhythmia, or death during the test. The 
risk of death for healthy adults who undergo maximum exercise is between 0 and 3 
deaths/100,000 tests. During the test, the participant’s heart rhythm will be constantly 
monitored by the study team. A participant will stop the test at the earliest sign of cardiac 
problems whether those signs are from the monitor or based on symptoms. Other risks of 
exercise include the risk of falls while using the treadmill or short-term myalgia or 
arthralgia caused by exercising that could last several days. Participants will be instructed 
to tell the study team if myalgia or arthralgia becomes bothersome as these may be 
treated with as needed mild pain relievers.
9.29
Risks of Qualitative Interviews
Answering questions about one’s illness could elicit negative feelings or emotions. 
Interviewers will be cognizant of this during interviewing and make every attempt to put 
the patient at ease and remind respondents that they can decline to answer questions 
and/or terminate the interview.
9.30
Blood measurements before and after CPET
There is minimal medical risk from blood sampling from the IV line.

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9.31
Risks of Effort-Expenditure for Rewards Task
This computer test of effort expenditure for perceived reward is similar to other computer 
based neuropsychological tests. This task is not harmful but may be frustrating or 
stressful. Participants may stop this task or withdraw from the study at any time and for 
any reason.
9.32
Risks of TMS
TMS may cause strong contractions of scalp muscles leading to discomfort or a 
headache. If participants find TMS too uncomfortable, they may stop it any time. 
Headaches usually go away by themselves or with nonprescription medication. The 
noise of the TMS magnet can damage hearing, so participants will be fitted with earplugs 
which must be worn during TMS. TMS can interfere with implanted medical devices.
Participants will not be able to have TMS if they have a pacemaker, implanted pump, a 
stimulator (such as a cochlear implant) or metal objects inside the eye or skull.
Risk of skin irritation at the site of the EMG electrode may be minimal in some subjects. 
There are no medical risks associated with surface EMG.
9.33
Risks of sleep electroencephalogram
There is no known risk concerning the EEG technique itself. The electrode cap can be 
uncomfortably tight. There may be mild irritation of the skin and scalp from the mild 
abrasion needed to assure good contact or from the collodion, paste, or gels used.
9.34
Risks of Medical, Psychiatric, Sleep Service and Rehabilitation 
Consultation
There is minimal medical risk or discomfort from medical, psychiatric, sleep, and 
rehabilitation consultation.
9.35
Risks of genetic testing
Genetic testing can provide information about how illness is passed on within a family. 
This knowledge may affect participants’ emotional wellbeing. They might feel differently 
about their life if they learned that they or their children were at increased risk of a 
disease, especially if there were no treatments. Their children, brothers or sisters may find 
out that they are at risk for health problems because of information found out about the 
participants, which might affect the participants’ relationships with family
members. Other family members may also be affected by uncovering risks they have but 
did not want to know about. This information can cause stress, anxiety, or depression.
Some genetic testing can also determine if people are directly related. These tests 
sometimes show that people were adopted or that their biological parent is someone other 
than their legal parent. If these facts were not known previously, they could be
troubling. Genetic counseling is available at the NIH to help participants understand the 
nature and implications of genetic findings.
Because of the emotional risk, some participants do not want to know the results of 
genetic testing. It is our policy to not disclose the results of genetic testing unless it may 
have direct medical or reproductive implications for participants or the participants’

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families. Any genetic results disclosed to participants will be done in the context of 
genetic counseling.
Results of genetic testing obtained at NIH are often preliminary and difficult to interpret 
because the testing is being done for research purposes only and the laboratories are not 
CLIA-certified. If a reportable genetic change is noted on a non—CLIA genetic test, the 
protocol will cover the costs of confirming the result using a CLIA genetic test.
Genetic information will be kept confidential to the extent possible. The results of genetic 
testing will be kept in a locked and secured manner at the NIH.
Problems, such as with insurance or employment discrimination, may occur if they 
disclose information about themselves or agree to have their research records 
released. We will not release any information about participants to any physician, 
insurance company or employer unless they sign a document allowing release of the 
information.
9.36
Risks of Skin Biopsies
The skin punch-biopsies have potential risks including infection, bleeding, pain, and 
potential scarring.
9.37
Risks of Muscle Biopsy
The muscle punch-biopsy procedure has potential risks including infection, bleeding, 
pain, and post-procedure anesthesia or paresthesia if a cutaneous nerve is damaged. The 
risk of these events is minimized by having the procedure performed only by experienced 
physicians under sterile conditions with adequate local anesthesia.
10 Subject Safety Monitoring
Study personnel, overseen by the PI, will closely monitor participant safety during study 
procedures. If, during the course of interviewing, assessment procedures and the research 
procedures, study staff identifies a condition that should require immediate clinical 
intervention, all necessary steps will be taken. Parameters to be monitored for safety 
reasons include level of consciousness. All MRI scans will be monitored in accordance 
with NIH Clinical Center policies. Toxicity will be assessed according to the most recent 
version of the NCI Common Terminology Criteria for Adverse Events (CTCAE).
Participants will be removed from the study if they are unable to cooperate with the study 
procedures, if a medical exclusionary condition develops, or if the PI judges 
discontinuation to be in the participant’s best medical interest.
11 Outcome Measures:
The primary purpose of this protocol is to perform exploratory analysis of collected 
samples for the generation of new hypotheses regarding ME/CFS. The types of analyses 
to be performed will be wide ranging. Planned areas of focus include:

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1.
Characterization of the immune system and inflammatory signaling in collected 
samples at baseline and following maximal exercise exertion.
2.
Characterization of the pattern of microbiome in collected samples at baseline and 
following maximal exercise exertion.
3.
Characterization of bioenergetics, autonomic, and metabolic function in collected 
samples at baseline and following maximal exercise exertion.
4.
Characterization of physical and cognitive fatigue using functional magnetic 
resonance imaging and transcranial magnetic stimulation at baseline and following 
maximal exercise exertion.
5.
Characterization of neurocognition at baseline and following maximal exercise 
exertion.
12 Statistical Analysis
12.1
Analysis of data/ study outcomes
The studies performed under this protocol are exploratory. Quantitative research data will 
be acquired and logged into a database. The PI or designated investigator will monitor 
data integrity. Because a variety of types of data will be obtained in the study, the 
statistical methods most appropriate for the nature of the data will be used.
Some of the relevant statistical approaches are as follows:
We are primarily interested in evaluating whether various types of data obtained are 
significantly different between the three groups as measured in cross-section during the 
phenotyping visit as well as between HV and PI-ME/CFS groups before and after an 
exercise stress. Each of these groups will be characterized using standard descriptive 
statistical methods.
As noted in Section 4.5, ME/CFS participants that are found no to meet the strict 
inclusion/exclusion of this study all have been diagnosed with ME/CFS by health 
providers and represent ME/CFS in the community. HVs and COVID-19 HVs that do 
not have fatigue or PEM may also be found to medical issues or other findings that 
would exclude them from meeting the strict inclusion/exclusion criteria of being 
healthy, but still represent a non-fatigue comparator to ME/CFS patients. The primary 
focus of the analyses detailed below will be between adjudicated ME/CFS participants 
and HVs and COVID-19 HVs that meet all inclusion/exclusion criteria. If the result of 
a primary analysis is positive, a secondary analysis will be repeated using all of 
participants. Findings that remain robust even when less stringent inclusion/exclusion 
criteria are used suggest that they are more likely to be replicable in community 
cohorts and perhaps worthy of further exploration.
For each continuous-scale outcome, repeated measures analysis of variance (ANOVA) 
will be used to test whether there are significant differences in cross-sectional 
measurements as well as in the pre and post measurements among two groups by using F 
tests. These initial tests will be performed using the adjudicated PI-ME/CFS group (i.e. 
participants determined to have PI-ME/CFS by the protocol’s adjudication process) 
compared to HV and COVID-19 HV participants meeting all inclusion and exclusion 
criteria. The cross-sectional analyses may then be repeated using all HV and COVID-19 
HV participants that did not report substantial clinical fatigue as noted above.

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If the overall multivariate test is significant, we will follow up with several post-hoc 
tests. Specifically, we will first conduct multivariate tests to compare the adjudicated PI- 
ME/CFS group (i.e. participants determined to have PI-ME/CFS by the protocol’s 
adjudication process) vs the other two control groups, and the PI-ME/CFS group vs each 
of the two control groups. These analyses will initially performed using only HV and 
COVID-19 HV participants meeting all inclusion and exclusion criteria. As noted above, 
for analyses that are statistically significant, the analysis will be repeated using all HV 
and COVID-19 HV participants that did not report substantial clinical fatigue to 
determine if the results remain statistically significant when less stringent 
inclusion/exclusion criteria are applied. 
Second, univariate ANOVA will be used for both cross-sectional measurements and pre 
and post exercise measurements separately to identify differences among groups. A

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similar univariate analysis will be conducted for the measurements of change from pre- to 
post exercise using the univariate ANOVA.
Additionally, descriptive statistics will be used to characterize the entire group of 
ME/CFS participants that completed the phenotyping evaluation regardless of 
adjudication. Comparisons will be made between the adjudicated PI-ME/CFS group and 
the phenotyping ME/CFS group. If there is no significant difference between the above 
two groups, it is not necessary to compare a ME/CFS group to our two control groups. 
Where statistically appropriate, the ANOVA methods described above will be used to 
compare the ME/CFS group with the two control groups.
Statistical approaches such as chi-square tests and multinomial logistic regression models 
will be used to examine the group differences for categorical outcomes.
A level of significance is set at 0.05. Multiple comparison test procedures such as 
Bonferroni and Dunnett’s methods will be used to examine the differences in each 
outcome measure among groups.
The primary MRI imaging aim is to test whether PI-ME/CFS group has significantly 
different connectivity patterns compared to the HV control group.
To this end, functional MRI measures will be taken at three conditions: 1) performing a 
force task, 2) performing a cognitive task, to define a network of interest, and 3) at rest to 
compute functional connectivity. At each condition, fMRI will be measured at two time 
points: pre exercise (preEx) and post exercise (postEx).
We will use a multivariate regression model where the response variable is connectivity 
strength among regions of interest pre- and post- exercise. Predictors include group status 
and fatigue scores.
Region of interest for the connectivity analysis will be defined based on tasks 1) and 
2). For this, data will be pre-processed with standard techniques using AFNI. Imaging 
quality assurance will be performed (i.e. motion correction parameters). We will fit a 
multivariate regression model to detect the differences in activity patterns of each task. 
Behavior and scales of fatigue will be entered as covariates.
Significance of the results will be set at p =0.05 corrected for multiple comparisons for all 
analyses.
Lastly, we will conduct receiver operating characteristic (ROC) analysis for selected 
markers that are potentially important, which will be identified by exploratory analysis 
and several statistical approaches mentioned above. We will evaluate the accuracy of 
each marker for its ability to classify subjects in the PI-ME/CFS group from those in the 
HV control group. For example, if MRI imaging measurements are significantly different 
among groups, next, we will calculate the nonparametric area under the ROC curve 
(AUC) for each imaging marker and its 95% confidence interval.

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12.2
Power analysis
The accrual ceiling for participants in the phenotyping and exercise stress arms of this 
study is 150 participants (50 per group) and dropouts will be replaced. The sample sizes 
were chosen based on convenience and equals about 1-2 participants per week. This is a 
phase I pilot study intended to explore a variety of clinical and biological data from the 
three different groups. For neuroimaging studies of somatosensory stimulation, it is 
estimated to require 12 subjects to achieve 80% power at the threshold of 0.05 to 
determine differences at the single voxel level for typical activations.114 The proposed 
sample size of 20 participants per group should be adequate for distinguishing group 
differences in fMRI activity during the proposed fatiguing tasks. The study is exploratory 
and its results will be used to estimate effect sizes to design a larger clinical study.
12.3
Analytic Plan
To analyze the vast, multidimensional data collected in this protocol, the research team 
has organized four Working Each Working Group has been assigned a post-
baccalaureate IRTA to assist in organization and with conducting a literature review.  
Groups to focus on four central themes: Clinical, Pathophysiology, Immunology, and 
Bioenergetics:
Clinical: Brian Walitt (Chair)
Data for analysis:

Life Narratives

Medical History

Physical Exam

Clinical laboratory measurements of blood and urine

Neuropsychological Testing data

SCID - 5

Patient Reported Outcome Measures

Polysymptom Interview

Activity Card Sort

Cardiopulmonary Exercise Test

Clinical Tilt Table Result

Clinical Sleep Study Result

Muscle Strength Grip Testing

Nutritional data

Microbiome

ME/CFS Criteria Adjudication
Pathophysiology: Mark Hallett (Chair)
Data for analysis: 

Neurological exams

Structural Brain MRI

Functional Brain MRI (Resting State, Diffuse Tensor Imaging, Hand Grip Task, Math 
Task)

Dynamic Tilt-table Study data

Single time point blood and CSF catecholamines measures

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
24 Hour Holter monitor data

Overnight sleep EEG data

Neuropsychological Testing data

Transcranial Magnetic Stimulation data

Salivary Cortisol measures (morning, afternoon, evening)

Patient Reported Outcome Measures

Cutaneous small fiber measurements of skin
Immunology: Avindra Nath and Steve Jacobson (Co-chairs)
Data for analysis: 

Clinical laboratory measures of inflammation

Brain MRI with gadolinium

Somalogic proteomics on blood and CSF

Flow cytometry on blood and CSF

RNASeq on PBMCs

GDF-15 measures

Muscle biopsy pathology (limited)

Muscle RNASeq

NK Cell functional assay

Tryptase 

Toxicity studies on PBMCs

Metabolomics of plasma and CSF

Iron levels of CSF

Exosomes of plasma

Neurofilament analysis of plasma and CSF
Bioenergetics: Kong Chen (Chair)
Data for analysis: 

Metabolic Chamber

Physical Activity Monitoring

DEXA measurement

Dietary History and Food Records

Cardiopulmonary Exercise Test

RNASeq on PBMCs

Seahorse mitochondrial assay on PBMCs

Muscle Strength Testing

Muscle biopsy pathology(limited)

Muscle RNASeq

Limited mitochondrial genetics data

Metabolomics of plasma and CSF

Microbiome
Each Working Group will begin by performing a literature review of their topic to 
ascertain the state of the ME/CFS field and guide their analyses. Analyses will be 
performed to both interrogate published findings to determine reproducibility and to 
make novel insights. Group members will first analyze the data that they are responsible 

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for collecting as part of the protocol. These individual data analyses will each be 
appropriate for the type of data collected and performed by Working Group members 
with experience in analyzing the data. Each Working Group will meet periodically to 
present their findings to each other, discuss issues of honing analyses, the need to expand 
data collection or perform additional experiments, identify areas of data consilience, and 
assemble a general consensus of how to interpret the results. 
Once each Working Group completes its analysis and evaluation of the data, a series of 
combined Working Group meetings will be convened. Each Working Group will present 
their methods and results to each other. Break-out sessions will be organized that will 
mix the members of the Working Groups together to discuss how findings in one domain 
may relate to those in other domains. An open discussion for all Working Group 
members will be conducted to discuss issues of honing analyses, the need to expand data 
collection or perform additional experiments, identify areas of data consilience, and 
assemble a general consensus of how to interpret the results. 
With the analyses of each Working Group completed, the research team will identify 
computational biologist collaborators to determine how to appropriately apply network 
analytics and machine learning approaches to the combined dataset. The goal of such 
analysis would to be elucidate observable linkages between the four research themes. 
These linkages would be areas of high interest in generating new hypotheses and also 
suggest potential therapeutic targets for future interventional studies. 
These Working Groups have already been initiated to perform an interim analysis on the 
ME/CFS and HV cohorts completed as of October 2020. 
13 Human Subjects Protection
13.1
Subject selection
Subject selection will be equitable among eligible participants. ME/CFS is reported to 
happen most commonly in persons aged 40-59.115 Increasing age also has significant 
effects of both on the experience of daily fatigue and on brain physiology. For this 
reason, individuals over the age of 60 will be excluded. Several of the neurocognitive 
instruments being used in this protocol require a 7th grade education for proper 
administration. These include the MMPI2, the Controlled Oral Word Association, and 
Grooved Pegboard tests. For this reason, persons with less than a 7th grade education will 
be excluded.
13.2
Justification for exclusion of children
Children less than 18 years old are excluded from the trial. Because ME/CFS is rare in 
children, pediatric cases may not be representative of the typical pathophysiology of the 
condition.
13.3
Justification for exclusion of pregnant women
Pregnancy-related physiological changes and fatigue would impact the interpretability of 
the results. Also, pregnancy is a contraindication for MRI. For these reasons, pregnant 
women are excluded from the study.

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13.4
Justification for exclusion of women who have given birth in the past 
year
Post-partum fatigue is likely to confound the results of the study.
13.5
Justification for exclusion of those who lack consent capacity
Persons who lack consent capacity will be excluded from this study. This study provides 
no direct benefit to the participant, involves varied and complex testing, and is more than 
minimal risk.

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Cognitive impairment severe enough to affect consent capacity would be unexpected in 
our target population. Such patients also would be unable to properly perform several 
study procedures, in particular patient-reported outcome scales and neurocognitive 
testing. For these reasons, persons with severe cognitive impairment will be excluded. If 
necessary, a CC-credentialed LIP will assess if a person is too cognitively impaired to 
participate in the protocol. While unexpected, a consultation to assess consent capacity 
will be obtained from the NIH Ability to Consent Assessment Team (301-496-9675) if 
necessary.
13.6
Justification of exclusion of non-English speakers
Persons who are not fluent in the English language will be excluded. Such persons would 
be unable to properly perform several study procedures that are only valid in the English 
language, in particular patient-reported outcome scales and neurocognitive testing.
13.7
Justification for inclusion of employees
NIH staff will not be solicited for participation, but will not be excluded if they express 
the desire to enroll. Protections for employees and staff participating in this study include
1) assuring that the participation or refusal to participate will have no effect, either 
beneficial or adverse, on the subject’s employment or position at NIH, 2) giving 
employees and staff who are interested in participating the “NIH Information Sheet on 
Employee Research Participation” prior to obtaining consent, and 3) assuring that there 
will be no direct solicitation of employees or staff. If the individual obtaining consent is a 
co-worker, an independent person (either from Bioethics or NIMH Human Subjects 
Protections Unit) will monitor the consent process for any NIH employees that desire to 
participate. Employees that participate in this protocol during work hours will be 
informed that they must obtain their supervisor’s permission and that per NIH guidelines 
they will not be paid.
13.8
Justification for sensitive procedures
Drug tapering is a sensitive procedure that may be performed on a subset of patients. 
Allowing for the use of central-acting medications would create problematic 
heterogeneity in the study groups. The impact of central-acting medications on biological 
measurements, in particular those made in blood, cerebrospinal fluid, fMRI, and 
neurocognitive testing, are also significant. As these medications do not treat the 
underlying cause of ME/CFS, do not substantially reduce morbidity or mortality, and 
have not been shown to lead to substantial clinical improvements, the period of tapering 
withdrawal will not likely to have substantial impact on health outcomes. As it is 
anticipated that a majority of participants will be taking at least one of these medications, 
it is necessary to allow for medication withdrawal rather than automatically excluding the 
potential participants. Participants taking stable doses of medications for medical diseases 
or psychiatric diagnoses will not be withdrawn from their medications. All withdrawal 
plans will require the agreement and full cooperation of the participant’s primary care 
physician. If psychiatric medications prescribed by the participant’s psychiatrist are being 
withdrawn, agreement and full cooperation from the psychiatrist will also be obtained.
Participants unable to tolerate drug tapering always have the option to remain or restart 
their medications and withdraw from the study.

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13.9
Safeguards for vulnerable populations and sensitive procedures 
Urine pregnancy testing will be performed on all persons of pregnancy potential at the 
time of admission for both Visit 1 and Visit 2. This pregnancy test will be considered 
valid for the entire period of the inpatient admission. Further, additional pregnancy
testing will be performed 24 hours prior to MRI scans performed in the NIH NMR center 
and prior to the use of ionizing radiation, such as DEXA, and fluoroscopy-guided LP.
Protections for employees and staff participating in this study include 1) assuring that the 
participation or refusal to participate will have no effect, either beneficial or adverse, on 
the subject’s employment or position at the NIH, 2) giving employees and staff who are 
interested in participating the “NIH Information Sheet on Employee Research 
Participation” prior to obtaining consent, and 3) assuring that there will be no direct 
solicitation of employees or staff. 4) Independent consent monitoring will be provided by 
the NIH HSPU or the Bioethics Department.
To ensure that research staff are adequately trained to respect the privacy and 
confidentiality of NIH employees and staff, study investigators and staff are required to 
complete the CITI “Just in Time” Training: Vulnerable Subjects – Workers/Employees.
This study collects sensitive information on drug use, alcohol use, and psychiatric 
diagnoses. The PI will train study staff regarding obtaining and handling potentially 
sensitive and private information about co-workers through staff discussions and written 
branch/section procedures. Information on drug and alcohol use will be in the 
participant’s NIH medical record.
To ensure that sensitive information collected as part of this protocol remain as private as 
possible under the law, a Certificate of Confidentiality was obtained from the Department 
of Health and Human Services (DHHS).
14 Anticipated Benefit: Indirect
This study does not offer direct benefit to participants but is likely to yield generalizable 
knowledge about ME/CFS.
15 Consent Documents and Process
The following section describes the informed consent process for participating in 
scientific protocol at NIH. The informed consent process for focus group participants is 
described fully in Section 4.3.
15.1
Consent procedures
All participants will receive a verbal explanation in terms suited to their comprehension 
of the purposes, procedures and potential risks of the study and of their rights as research 
participants. Participants will have the opportunity to carefully review the written consent 
form and ask questions regarding this study prior to signing.

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Participants who have completed participation (prior to Amendment 11) will be offered 
the opportunity to complete missing research procedures and undertake optional 
procedures made available in prior Amendments. These individuals will complete a 
written re-consenting process with the most recent version of the informed consent form. 
Persons who have completed participation (prior to approval of Amendment I) will also 
be offered the option of undergoing whole genome sequencing. These individuals will be 
contacted by email or telephone to determine if they are interested. Interested participants 
will be mailed a copy of the whole genome sequencing informed consent form. The 
individual will be instructed to read the consent form and be ready at the agreed upon 
date and time to review the consent document with an investigator authorized to obtain 
consent.
The consenting investigator will document the date and time oral consent was obtained 
on a consent procedure note. The participant will sign and date the consent form in the 
presence of a witness. The witness will also sign and date the consent form. The subject 
will be instructed to mail the consent form back to the site, where the investigator will 
sign and date the form. The investigator will then send a copy of the form with all 
signatures to the subject. The original consent document will be placed in the research 
chart.
15.2
Consent documents
The consent form contains all required elements. One consent form will be used for 
ME/CFS and HV participants in the main study (ME/CFS, HV Consent Form). A 
separate consent form will be used for COVID-19 HV participants in the main study 
(COVID-19 HV Consent Form). Two separate consent forms will be used for 
participants in Focus Group A and for Focus Group B. A separate consent form will be 
used for participants in the technical development sub-study (Technical Development 
Substudy Consent Form). A separate consent for will be used for obtaining consent for 
whole genome sequencing (Whole Genome Sequencing Consent Form).
16 Data and Safety Monitoring
16.1
Data and safety monitor
Data and safety will be monitored by the Principal investigator in conjunction with an 
independent medical monitor. The independent medical monitor is Dr. Camilo Toro.
16.2
Data and safety monitoring plan
Study investigators will evaluate the safety of study subjects throughout the conduct of 
the study and respond to adverse events (AEs) in a timely manner. The PI in 
collaboration with associate investigators will review any cases associated with serious 
adverse outcomes and advise on whether or not any changes in the research plan are 
warranted. Data and safety monitoring will occur annually,

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16.3
Criteria for stopping the study or suspending enrollment or 
procedures
If, in the judgment of the PI and the adjunct PI, a specific study procedure is yielding 
frequent unexpected or adverse outcomes, that procedure will be suspended until a 
review can be undertaken in consultation with the IRB. Depending on that consultation, 
the procedure may be dropped from the protocol via an amendment, or specific language 
may be added to the protocol and consent forms to reflect the changing risk level.
17 Quality Assurance
17.1
Quality assurance monitor
The NINDS Quality Assurance Audit Committee will monitor the study.
17.2
Quality assurance plan
This protocol will undergo periodic review by the NINDS Quality Assurance (QA) Audit 
Committee as outlined in the NINDS QA Standard Operating Procedure. The purpose of 
the QA audit is to assess compliance with applicable regulatory requirements, good 
clinical practice guidelines, NINDS policy, as well as to provide recommendations for 
improving the management of clinical research data. The protocol will be audited a 
minimum of one time a year.
18 Reporting of Unanticipated Problems, Adverse Events and Protocol Deviations
Reportable events for this protocol will be tracked and reported in compliance with 
policy 801.
19 Alternatives to Participation
Subjects do not receive any treatment in this study or forego any treatment in order to 
participate in this study. The alternative, therefore, is not to participate.”
20 Privacy
All research activities will be conducted in as private a setting as possible.
21 Confidentiality
21.1
For research data and investigator medical records
Each participant will receive a unique study ID number at the time of enrollment. A 
master list correlating the unique numbers with personal health information such as 
names, addresses, and contact information will exist but will only be accessible to 
investigators on this protocol in order to minimize the risk of compromising 
confidentiality. These data will be kept in password-protected computers on the NIH 
campus.
The results of the research tests will not be shared with participants, other than those that 
are important for the participant’s health and well-being. Clinically-relevant information 
will be shared with the participant and the participant’s physician, with the participant’s

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permission or upon request. Genetic results will be confirmed at NIH expense in CLIA 
lab prior to returning to participants or providers as detailed in Section 9.35.
21.2
For stored samples
Data and samples will be stored in secured areas. Samples kept at the NIH will be stored 
in a coded fashion in a locked room in the laboratories of the study investigators.
21.3
Special precautions
Samples and data will be stored using codes that we assign. Data will be kept in 
password-protected computers. Samples will be kept in locked storage. Only study 
investigators will have access to the samples and data.
22 Conflict of Interest
22.1
Distribution of NIH guidelines
NIH guidelines on conflict of interest have been distributed to all investigators.
22.2
Conflict of interest
There are no conflicts-of-interest to report.
23 Technology Transfer
No technology transfer agreements are needed for this study.
24 Research and Travel Compensation
Participants will be compensated for research-related inconveniences. Compensation will 
be prorated for parts completed if subjects do not complete the study. Payments will be 
sent after each visit. The schedule for procedure-based payments is below:
Phenotyping Visit

$10 for physical exam

$20 for IV line

$20 for blood draw

$10 for a urine sample

$10 for symptom questionnaires (13)

$10 for the mental health assessment/SCID

$10 for psychiatric inventories (10)

$25 for neurocognitive testing

$25 for occupational therapy evaluation

$50 for the structural brain MRI scan

$30 for MRI contrast

$10 for muscle strength testing

$10 for activity level monitoring training

$10 for nutritional assessment

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
$10 for saliva collection

$10 for buccal swab

$10 for stool sample

$20 for autonomic testing

$200 for lumbar puncture

$2-$8.60 for each Effort for Reward Task (1)

Immune cell collection:

(Option A): $110 for cytapheresis (2 pass)

(Option B): $60 for immune cell blood draw
Exercise Stress Visit

$10 for physical exam

$20 for IV line

$20 for admission blood draw

$10 for a urine sample

$10 for saliva collection (2)

$30 for stool samples

$10 for admission questionnaires

$10 for activity level monitoring

$25 for DEXA

$50 for exercise testing (CPET)

$100 for CPET blood draws (4-6)

$10 for CPET qualitative interviews (4-6)

$10 for CPET symptom questionnaires (4-6)

$10 for CPET neurocognitive testing

$20 for Holter monitor

$2-$8.60 for each Effort for Reward Task (2)

$70 for each fMRI scan session (2)

$200 for lumbar puncture

$100 for sleep electroencephalograms

Resting energy expenditure measurements:

(Option 1): $250 for inpatient stay in metabolic chamber and one ventilated hood 
measurement

(Option 2): $40 for ventilated hood measurements (4-6)
Optional Procedures

$100 for the skin biopsies

$250 for a muscle biopsy

$70 for fMRI scan session

$50 for each transcranial magnetic stimulation session

$150 for sleep electroencephalograms + Metabolic Chamber + DEXA

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Compensation for completing the phenotyping visit for PI-ME/CFS and HV participants 
is $500. Compensation for completing the phenotyping visit for persons choosing to have 
cytapheresis will be $610. Compensation for completing the phenotyping visit for 
persons choosing to have immune cell blood draw will be $560.
Compensation for completing the phenotyping visit for DLI-A participants is $465.
Compensation for completing all procedures during the exercise stress visit will be $915. 
Participants receiving ventilated hood measurements in lieu of staying in the metabolic 
chamber will receive $705.
The total compensation for completing the protocol will be $1,415. Maximum 
compensation for participation in additional procedures will be $1,795. Participants may 
earn up to an addition $6.00 - $25.80 if they complete the Effort for Reward Task three 
times.
Compensation for the optional skin biopsies will be $100. Compensation for the optional 
muscle biopsy will be $250. Compensation for the optional fMRI session will be $70.
Compensation for the optional TMS session will be $50. Compensation for the optional 
sleep electroencephalogram + Metabolic Chamber + DEXA will be $150. Participants 
undergoing additional immune cell collections will be compensated ($110 for 
cytaphersis, $60 for large volume blood draw).
Compensation for travel and lodging will be provided for each participant. Travel, 
lodging and meal vouchers will be provided for a single companion for ME/CFS 
participants. Only local travel will be compensated for participants in the technical 
development sub-study.
Focus group participants will not be compensated.
The schedule for payment for participants in the technical development sub-study is 
described below:

$10 for each hour of time (up to 8 hours)

$40 for fMRI scan session

$40 for transcranial magnetic stimulation session

$20 for EEG testing

$10 for questionnaires
The maximum compensation for technical development participants that complete both 
the TMS and fMRI tasks is $190. The payment for the technical development study was 
increased since the start of the protocol. Retrospective payments were made to these 
participants. A letter to this effect is detailed in Appendix 26.9.

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NIH employees and staff who participate during work hours must have permission from 
their supervisor. NIH employees must either participate outside of work hours or take 
leave in order to receive compensation.

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25 Attachments/ Appendices

ME/CFS at NIH
25.1
Schedule of Events
Study Overview: PI-ME/CFS, Healthy Volunteers
Procedures
Pre-Screening
Phenotyping Visit 
(V1)
Between Visits
Exercise Stress Visit 
(V2)
Telephone Contact
X
X
Obtain Medical Records
X
Contact Participant Primary 
Physician
X
X
Informed Consent
X
History and Physical Exam
X
X
Intravenous Line
X
X
Screening Blood Assessment
X
X
Urine Collection
X
X
Symptom Assessment
X
X
Psychological Assessment
X
Neurocognitive Testing
X
X
Occupational Therapy Evaluation
Only PI-ME/CFS
Clinical Brain MRI
X
Muscle Strength Testing
X
Activity Monitoring/Fatigue Diary
Instructions 
Given
X
X
Nutritional Assessment
X
X
X
Saliva Sample Collection
X
X
Buccal Swab Sample Collection
X
Stool Sample Collection
X
X
Holter Monitoring
X
X
Lumbar Puncture
X
X
Autonomic Testing
Optional
Immune Cell Collection
X
Optional
Medication Review/Wash-out 
planning
X
Medication Wash-out Monitoring
X
X
Clinical Center Consultation
Optional
Optional
Neuropsychological Measurements
X
X
Case Adjudication
X
Metabolic Chamber
Optional with 
Sleep EEG
Optional with 
Sleep EEG
X
Ventilated Hood Measurements
X
Dual Energy X-ray Absorptiometry
Optional with 
Sleep EEG
Optional with 
Sleep EEG
If not previously 
completed
Peak Exercise Testing
X
Qualitative Measurement of PEM
X
Functional Magnetic Resonance 
Imaging
Optional
Optional
X
Transcranial Magnetic Stimulation
Optional
Optional
Optional
Effort to Reward Task
X
X
Sleep EEG
Optional
Optional
X
Skin Biopsy
Optional
Optional
Optional
Muscle Biopsy
Optional
Optional
Optional
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Study Overview: COVID-19 Healthy Volunteers
Procedures
Pre-Screening
Phenotyping Visit 
(V1)
Telephone Contact
X
Obtain Medical Records
X
Contact Participant Primary 
Physician
X
Informed Consent
X
History and Physical Exam
X
Intravenous Line
X
Screening Blood Assessment
X
Urine Collection
X
Symptom Assessment
X
Psychological Assessment
X
Neurocognitive Testing
X
Clinical Brain MRI
X
Muscle Strength Testing
X
Nutritional Assessment
X
Saliva Sample Collection
X
Buccal Swab Sample Collection
X
Stool Sample Collection
X
Holter Monitoring
X
Lumbar Puncture
X
Autonomic Testing
Optional
Immune Cell Collection
X
Neuropsychological Measurements
X
Effort to Reward Task
X
Skin Biopsy
Optional
Muscle Biopsy
Optional
Functional MRI
Optional
Transcranial Magnetic Stimulation
Optional

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Exercise Stress Visit Overview
Procedures
Pre-Exercise
Exercise Day 0
Post-Exercise Days 1-4
Informed Consent Review
X
History and Physical Exam Update
X
Intravenous Line
X
Blood Draw
Optional
Urine Collection
X
X
X
Saliva Collection
X
X
Stool Sample Collection
X
X
X
Symptom Assessment
X
X
X
Activity Monitoring/Fatigue Diary
X
X
X
Nutritional Assessment
X
Metabolic Diet
X
X
X
Neurocognitive Testing
X
X
Metabolic Chamber
X
X
X
Ventilated Hood Measurements
X
Optional
Dual Energy X-ray Absorptiometry
If not performed previously
Holter Monitoring
X
X
Peak Exercise Testing
X
Functional Magnetic Resonance 
Imaging
X
X
Effort to Reward Task
X
X
Serial Qualitative Interviews of 
PEM
X
Serial Blood Draws
X
X
Lumbar Puncture
X
Sleep EEG
X
X
X
Clinical Center Consultation
Optional
Optional
Optional
Skin Biopsy
Optional
Optional
Muscle Biopsy
Optional

ME/CFS at NIH
25.2
Questionnaires
Attached in PTMS.
25.3
Case Report Forms (CRFs)
Clinical data will be captured using the NIH Clinical Research Information System 
(CRIS) as is required per NIH policy. We will work with CTDB to develop forms to 
capture all required research data.
25.4
Focus Group Forms
All materials required to recruit, screen, and perform the Focus Group interviews are 
available in PTMS.
25.5
Advertisements
Flyers and Public Service Announcements have previously been approved. There are 
three flyers (ME/CFS, COVID-19 HV, and HV) and three PSAs (ME/CFS, COVID-
19 HV, and HV).
25.6
Website Language
The most current version of the website language is attached in PTMS.
25.7
Screening Questionnaires
The protocol’s screening questionnaire for Patient Recruitment Office has been 
previously approved.
25.8
Thank you letters
Thank you letters will be sent to each participant on the completion of their participation. 
A thank you letter for participating in the main study and the focus groups study are 
attached.
25.9
Letter for Retroactive Increase in Compensation for Technical 
Development Substudy
A letter will be sent to prior participants that completed the technical development 
substudy to inform that they will be receiving additional compensation for their 
involvement.
25.10
Adjudication Board Members
The list of the current members of the Adjudication Board has been previously approved.
26 Consent Forms
Al consents attached in PTMS: 
ME/CFS, HV Consent Form 
COVID-19 HV Consent Form
Focus Group Study A Consent Form 
Focus Group Study B Consent Form
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Technical Developments Consent Form 
Genetic Consent Form
27 References
1.
AN OUTBREAK of encephalomyelitis in the Royal Free Hospital Group, 
London, in 1955. British medical journal. 1957;2(5050):895-904.
2.
Acheson ED. Benign myalgic encephalomyelitis. Lancet. 1957;272(6973):834- 
835.
3.
Holmes GP, Kaplan JE, Gantz NM, et al. Chronic fatigue syndrome: a working 
case definition. Annals of internal medicine. 1988;108(3):387-389.
4.
Epidemic myalgic encephalomyelitis. British medical journal.
1978;1(6125):1436-1437.
5.
Wessely S. Old wine in new bottles: neurasthenia and 'ME'. Psychological 
medicine. 1990;20(1):35-53.
6.
McEvedy CP, Beard AW. Concept of benign myalgic encephalomyelitis. British 
medical journal. 1970;1(5687):11-15.
7.
O'Hern EM. Alice Evans and the brucellosis story. Annali Sclavo; rivista di 
microbiologia e di immunologia. 1977;19(1):12-19.
8.
Gilliam AG. Epidemiological study on an epidemic, diagnosed as poliomyelitis, 
occuring among the personnel of Los Angeles County General Hospital during 
the summer of 1934. In: Department UST, ed. Vol 240. Washington DC: United 
States Treasury Department Public Health Service Public Health Bulletin; 1938:1- 
90.
9.
Jones JF, Ray CG, Minnich LL, Hicks MJ, Kibler R, Lucas DO. Evidence for 
active Epstein-Barr virus infection in patients with persistent, unexplained 
illnesses: elevated anti-early antigen antibodies. Annals of internal medicine. 
1985;102(1):1-7.
10.
DeFreitas E, Hilliard B, Cheney PR, et al. Retroviral sequences related to human 
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