Conditions: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Chronic Fatigue Syndrome Myalgic Encephalomyelitis Exercise Intolerance Dysautonomia Low Ventricular Filling Pressures (Preload Failure) Postural Orthostatic Tachycardia Syndrome Orthostatic Hypotension Fibromyalgia
Keywords: Myalgic encephalomyelitis/Chronic fatigue syndrome Myalgic encephalomyelitis Chronic fatigue syndrome Pyridostigmine Exercise Intolerance Invasive Cardiopulmonary Exercise Test
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 45
Healthy volunteers: no

Brief summary:
Myalgic encephalomyelitis/Chronic fatigue syndrome (ME/CFS), otherwise known as Chronic fatigue syndrome (CFS) or myalgic encephalomyelitis (ME), is an under-recognized disorder whose cause is not yet understood. Suggested theories behind the pathophysiology of this condition include autoimmune causes, an inciting viral illness, and a dysfunctional autonomic nervous system caused by a small fiber polyneuropathy. Symptoms include fatigue, cognitive impairments, gastrointestinal changes, exertional dyspnea, and post-exertional malaise. The latter two symptoms are caused in part by abnormal cardiopulmonary hemodynamics during exercise thought to be due to a small fiber polyneuropathy. This manifests as low biventricular filling pressures throughout exercise seen in patients undergoing an invasive cardiopulmonary exercise test (iCPET) along with small nerve fiber atrophy seen on skin biopsy.

After diagnosis, patients are often treated with pyridostigmine (off-label use of this medication) to enhance cholinergic stimulation of norepinephrine release at the post-ganglionic synapse. This is thought to improve venoconstriction at the site of exercising muscles, leading to improved return of blood to the heart and increasing filling of the heart to more appropriate levels during peak exercise. Retrospective studies have shown that noninvasive measurements of exercise capacity, such as oxygen uptake, end-tidal carbon dioxide, and ventilatory efficiency, improve after treatment with pyridostigmine. To date, there are no studies that assess invasive hemodynamics after pyridostigmine administration.

It is estimated that four million people suffer from ME/CFS worldwide, a number that is thought to be a gross underestimate of disease prevalence. However, despite its potential for debilitating symptoms, loss of productivity, and worldwide burden, the pathophysiology behind ME/CFS remains unknown and its treatment unclear. By evaluating the exercise response to cholinergic stimulation, this study will shed further light on the link between the autonomic nervous system and cardiopulmonary hemodynamics, potentially leading to new therapeutic targets.

Detailed description:
The hypothesis of our study is that hemodynamic, ventilatory and oxygen exchange variables such biventricular filling pressures and systemic oxygen extraction can be improved by cholinergic stimulation in patients with ME/CFS.

The objective of this study is to examine the exercise response to pharmacologic cholinergic stimulation in ME/CFS patients already undergoing a clinically indicated invasive cardiopulmonary exercise test (iCPET). This will be achieved by inhibiting acetylcholinesterase with pyridostigmine, thus increasing acetylcholine levels, downstream levels of norepinephrine, and enhancing vascular regulation.

To test our hypothesis, we propose the following specific aims:

Define the response of peak oxygen uptake(VO2) to pyridostigmine. Define the gas exchange responses, such as end-tidal carbon dioxide(CO2) and ventilatory efficiency to pyridostigmine.

Define the hemodynamic responses, such as right atrial pressures, pulmonary artery pressure, pulmonary capillary wedge pressures, cardiac output, heart rate, stroke volume, pulmonary vascular resistance and systemic vascular resistance to pyridostigmine.

Evaluate the response of skeletal muscle oxygen extraction and lactate to pyridostigmine.

These determinations will occur during a clinically indicated iCPET, which includes exercising on a stationary cycle with a right heart catheter (RHC) and a radial arterial line in place. To stimulate the cholinergic response, a single dose of an oral acetylcholinesterase inhibitor, pyridostigmine, versus placebo will be given after the iCPET. Recovery cycling will be performed after a rest period of 50 minutes. This will be administered in a randomized, double-blind, placebo-controlled trial.

Arms:
arm 1: Pyridostigmine 60 mg by mouth as a one time dose arm 2: Placebo by mouth as a one time dose

Interventions: intervention 1: Pyridostigmine Bromide intervention 2: Placebo
Intervention descriptions: intervention 1: Pyridostigmine Bromide 60 mg capsule by mouth as a one time dose intervention 2: Placebo (Cellulose microcrystalline) capsule by mouth as a one time dose

Protocol document:
Prot_SAP_000.pdf: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The Exercise Response to Pharmacologic Cholinergic Stimulation in Myalgic 
Encephalomyelitis/Chronic Fatigue Syndrome – Detailed Protocol 
NCT #: 03674541 
Date: September 3, 2020 


 
1 
Detailed Protocol 2018P001871 
  
Protocol Title: The Exercise Response to Pharmacologic Cholinergic Stimulation in 
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome 
  
Principal Investigator: David Systrom, MD;  
  
Protocol Version Number: 06 
  
Date: 9/3/2020 
  
I. Background and Significance 
  
Myalgic encephalomyelitis/Chronic fatigue syndrome (ME/CFS), otherwise known as 
Chronic fatigue syndrome (CFS) or myalgic encephalomyelitis (ME), is an under-
recognized disorder whose cause is not yet understood. It is characterized by a 
constellation of symptoms that include debilitating fatigue for at least six months, post-
exertional 
malaise, 
exertional 
dyspnea, 
cognitive 
impairment, 
orthostatic 
intolerance/autonomic dysfunction, widespread pain, gastrointestinal motility 
disorders, and unrefreshing sleep. Depending on varying definitions, ME/CFS is 
estimated to range from 10 to 25 percent of patients seen in primary care practices,1 
75 to 267 cases per 100,000 persons,2 or 836,000 to 2.5 million people in the United 
States.3 In the United States, the lost productivity due to this disease results in an 
annual loss of $20,000 per person with CFS, or $9.1 billion.4 Because of the often 
disconnected symptoms and resulting unnecessary medical tests and procedures, this 
number is thought to be an underestimation and may actually approach $23 billion in 
direct and indirect costs to society.5 
  
The Institute of Medicine has published diagnostic criteria for ME/CFS.3 Major criteria 
include substantial impairment from fatigue for six months, post-exertional malaise, 
and unrefreshing sleep. Minor criteria include cognitive impairment and orthostatic 
intolerance. As stated above, the cause of the syndrome is unknown with proposed 
mechanisms related to viral or infectious causes, immune system dysfunction, 
endocrine disorders, and neural dysregulation/autonomic dysfunction. Mounting 
evidence suggests that dysautonomia/autonomic dysfunction plays a prominent role 
in the pathophysiology of ME/CFS. Studies have shown an overlap in symptoms and 
diagnoses between postural orthostatic tachycardia syndrome (POTS), fibromyalgia, 
and ME/CFS.6,7 Additionally, recent data have shown a high prevalence of a small 
fiber neuropathy (SFN) seen on skin biopsy in patients with chronic widespread pain 
syndromes,8 fibromyalgia,9 and POTS.10 
  
Because of the significant exertional symptoms these patients suffer from, the Institute 
of Medicine and others have proposed changing the name of ME/CFS to Systemic 
Exertion Intolerance Syndrome.11 Patients with SFN and unexplained dyspnea make 
up a prominent proportion of the patients seen in the Dyspnea Center at Brigham and 
Women’s Hospital. These patients often go years without a diagnosis before an 
evaluation by a pulmonary physician at the Dyspnea Center. They are characterized 
as otherwise healthy individuals with diagnoses of ME/CFS, POTS, orthostatic 
hypotension, and/or fibromyalgia. Symptoms include exertional dyspnea, post-


 
2 
exertional malaise, pre-syncope, and perioral numbness and tingling due to 
hyperventilation. 
  
A significant number of these patients have been evaluated clinically with a level 3 
cardiopulmonary exercise tests (CPET). This involves the placement of a pulmonary 
artery catheter and radial artery catheter in the cardiac catheterization suite. After 
these are placed and secured, the patient performs upright exercise on a stationary 
cycle ergometer against increasing resistance using a ramp protocol.12 Simultaneous 
measurements of pressures in three chambers of the heart (right atrium, right ventricle, 
pulmonary capillary wedge pressure/left atrial pressure/left ventricular end diastolic 
pressure), the pulmonary artery pressure, mean arterial pressure, and gas exchange 
variables (oxygen uptake and carbon dioxide production) are obtained from the resting 
state to the post-exercise state. Blood gas samples are drawn from both the arterial 
and venous compartment throughout exercise to analyze oxygen extraction, pH, 
lactate, partial pressure of oxygen (O2), and the partial pressure of carbon dioxide 
(CO2). Using the Fick principle, cardiac output is calculated, and various 
cardiopulmonary diseases are ruled out based on the combination of oxygen uptake, 
cardiac output, peripheral oxygen extraction, and cardiac chamber pressure 
measurements. 
  
Our previous study has shown that low biventricular filling pressures (“preload failure”, 
PLF) seen during level 3 CPET is the cause of unexplained dyspnea in patients with 
ME/CFS.13 In this study, impaired patients, defined as oxygen uptake below 80% of 
predicted, were compared to normal patients after the exclusion of heart failure, 
pulmonary hypertension, and pulmonary mechanical limits to exercise. These data 
show that subjects in the impaired group have significantly lower and reversible 
biventricular filling pressures at peak exercise, suggesting this as a cause of lower 
oxygen uptake and decreased cardiac output. Of the impaired subjects who 
underwent evaluation for underlying causes, a significant number had positive findings 
for dysautonomia either by tilt table testing or nerve conduction studies. 
  
As mentioned above, numerous studies have demonstrated up to an approximately 
fifty percent prevalence of SFN in chronic widespread pain, fibromyalgia, and POTS. 
Studies have shown that in POTS, there is abnormal venous pooling in the legs upon 
standing.14 Furthermore, these patients demonstrate a hypersensitivity to 
norepinephrine or phenylephrine infusions in relation to peripheral venoconstriction, 
suggesting that they have an upregulation of adrenergic receptors in response to 
nerve atrophy and a lack of endogenous catecholamine production.15,16 This is 
reinforced by the observation of decreased norepinephrine spillover after sympathetic 
nervous system evaluation in patients with POTS.17 
  
Because of the evidence of impaired neural regulation of venous tone as a cause of 
symptoms in POTS, therapies aimed at increasing venoconstriction or intravascular 
volume have been attempted with varying degrees of success. These include 
midodrine, salt tabs, aggressive oral hydration, and fludrocortisone. Augmenting 
neurotransmitter release of norepinephrine with pyridostigmine has been studied with 
significant improvement in both symptom burden and heart rate response.18 By 
inhibiting acetylcholinesterase, pyridostigmine is thought to enhance cholinergic 
stimulation of norepinephrine release at the post-ganglionic synapse.18 
 


 
3 
 
  
Recent work analyzing the Brigham and Women’s Hospital CPET database has 
shown that among patients with low filling pressures (” preload failure”), approximately 
50% has SFN seen on skin biopsy (data unpublished, manuscript in process). Off-
label use of pyridostigmine for treatment of preload failure has shown a significant 
increase either in submaximum19 or maximum aerobic capacity.20 This is thought to 
be due to the same mechanisms described above; increased catecholamine release 
results in improved venoconstriction during exercise, thus increasing venous return to 
the heart. 
  
While retrospective reviews have been published about SFN and dysautonomia, 
prospective treatment studies are scarce, and none has been published about the 
treatment of preload failure. In the past, dysautonomia and preload failure account for 
up to 20% of the diagnoses obtained from the CPET program21, but more recently the 
prevalence is as high as 50%. The aim of this study is to assess the exercise response 
to pharmacologic cholinergic stimulation with pyridostigmine in preload failure. This 
will be evaluated in a randomized, double-blind, placebo-controlled trial. ME/CFS is 
remains a disease of unclear etiology with a significant worldwide burden. This study 
will enhance our knowledge into the pathophysiology behind ME/CFS and create new 
treatment strategies. 
  
II. Specific Aims 
  
The hypothesis of our study is that hemodynamic, ventilatory and oxygen exchange 
variables such biventricular filling pressures and systemic oxygen extraction can be 
improved by cholinergic stimulation in patients with ME/CFS.  
 
The objective of this study is to examine the exercise response to pharmacologic 
cholinergic stimulation in ME/CFS patients already undergoing a clinically indicated 
level 3 cardiopulmonary exercise test (CPET). This will be achieved by inhibiting 
acetylcholinesterase with pyridostigmine, thus increasing acetylcholine levels, 
downstream levels of norepinephrine, and enhancing vascular regulation. 
  
To test our hypothesis, we propose the following specific aims: 
  
Primary Aim: 
 


 
4 
1. Define the response of peak oxygen uptake (VO2) to pyridostigmine 
 
Secondary Aims: 
 
1. Define the gas exchange responses, such as end-tidal CO2 and ventilatory 
efficiency to pyridostigmine. 
2. Define the hemodynamic responses, such as right atrial pressures, pulmonary 
artery pressure, pulmonary capillary wedge pressures, cardiac output, heart 
rate, stroke volume, pulmonary vascular resistance and systemic vascular 
resistance to pyridostigmine. 
3. Evaluate the response of skeletal muscle oxygen extraction and lactate to 
pyridostigmine.          
 
 
These determinations will occur during a clinically indicated level 3 CPET, which 
includes exercising on a stationary cycle with a right heart catheter (RHC) and a radial 
arterial line in place. To stimulate the cholinergic response, a single dose of an oral 
acetylcholinesterase inhibitor, pyridostigmine, versus placebo will be given after the 
level 3 CPET. Recovery cycling will be performed after a rest period of 50 minutes. 
This will be administered in a randomized, double-blind, placebo-controlled trial. 
  
The above variables have been well described as objective markers of exercise 
capacity and its use in diagnosing cardiopulmonary diseases.22 Many of these 
variables are based on the Fick principle: 
  
Oxygen uptake = Cardiac Output *(Arterial-venous oxygen content difference) 
  
Oxygen uptake (VO2) and carbon dioxide output (VCO2) are continuously measured 
using a metabolic cart. Based on the equation above, oxygen uptake represents a 
composite of cardiac output and peripheral oxygen extraction, thus revealing the 
status of the cardiovascular system as a whole. This is further analyzed by obtaining 
both arterial and mixed venous oxygen saturations during exercise through the radial 
and pulmonary artery catheters, allowing a calculation of the arterial-venous oxygen 
content difference. Finally, oxygen uptake and the arterial-venous oxygen content 
difference is used to calculate cardiac output using the Fick principle outlined above. 
  
The arterial-venous oxygen content difference is used to assess peripheral oxygen 
extraction. By adjusting this to the patient’s hemoglobin, we can evaluate for 
macroscopic shunts, peripheral shunts, and mitochondrial dysfunction. 
  
Ventilatory efficiency is defined as Minute Ventilation (VE)/Carbon Dioxide production 
(VCO2). Through the alveolar ventilation equation (see below), we are able to 
determine if there is increased dead space ventilation or hyperventilation. Additionally, 
end-tidal CO2 measurements will be used to determine if hyperventilation is present. 
 
VA = 
!"#$%&
'()*$    ;     𝑉𝐴= 𝑅(𝑉𝑇−𝑉𝐷) 
 
 Where 
VA: alveolar ventilation (mL/min) 
VCO2: Rare of CO2 production (mL/min) 
PACO2: Alveolar PCO2 (mmHg) 


 
5 
R: Respiratory rate (breaths/min) 
K: Constant (863 mmHg) 
VT: Tidal volume (mL) 
VD: Physiologic dead space (mL) 
 
 
Right atrial pressures and pulmonary capillary wedge pressure measurements are 
obtained through the pulmonary artery catheter and measured with the Philips Xper 
Information Management System. Normal values and tracings have been well 
described in the past and the physician investigators are well versed in interpreting 
these values and waveforms.23 Based on prior studies, a right atrial pressure of 6.5 
mmHg will be considered the lower limit of normal at peak exercise.13 
  
III. Subject Selection 
  
  
All adults between 18 and 80 years old who are referred for a clinically indicated level 
3 CPET (stationary cycle exercise testing with a RHC and radial arterial line place) for 
the evaluation of unexplained exercise intolerance, most often from BWH outpatient 
pulmonary and cardiology clinics will be pre-screened for this study. Prospective 
subjects’s medical records will be initially pre-screened for the following initial eligibility 
criteria: 
  
Initial (Pre-screening) Inclusion criteria: 
  
1) Meets the Institute of Medicine (IOM) criteria for ME/CFS, or 
2) Has medical comorbidities associated with ME/CFS such as dysautonomia, low 
ventricular filling pressures, postural orthostatic tachycardia syndrome (POTS), 
orthostatic hypotension, or fibromyalgia.  Further confirmation that a subject 
meets the IOM criteria for ME/CFS by the telephone pre-screening questionnaire 
will be needed for subjects that meet this Initial Pre-screening inclusion criteria. 
 
Initial (Pre-screening) Exclusion criteria: 
  
1) Obesity (BMI > 30 kg/m2) 
2) Non-controlled asthma 
3) Anemia (Hb < 10 g/dl) 
4) Active or treated cancer 
5) History of interstitial lung disease (ILD) 
6) Chronic obstructive pulmonary disease (COPD) 
7) Pulmonary hypertension (PH) 
8) Congestive heart failure (CHF) 
9) Active arrhythmias 
10) Valvular heart disease 
11) Coronary artery disease (CAD) 
12) Other conditions that could predict a limitation or not completion of the study (as 
determined by the PI). 
  
  


 
6 
Based on the medical history obtained from the electronic medical record, potential 
subjects will receive a recruitment letter at least one week prior to their CPET, and if 
a potential subject is interested in the study, a consent form will be given no less than 
two days before their test.  
  
On the date of their test, potential subjects, who agree to participate and sign the 
consent form, will be evaluated for the following final eligibility criteria. 
  
Final (Screening) Inclusion criteria: 
  
1) Completing the clinically indicated level 3 CPET 
  
Final (Screening) Exclusion criteria: 
  
1) Pregnancy test positive in female patients. 
2) Submaximal testing in clinically indicated level 3 CPET: peak HR < 80% predicted 
OR peak RER < 1.05. 24 
3) Pulmonary mechanical limitation to exercise in clinically indicated level 3 CPET: 
VE /MVV < 0.7 at AT.25 
4) Pulmonary arterial hypertension in clinically indicated level 3 CPET rest mPAP > 
25 mmHg and rest PCWP < 15 mmHg and PVR ≥ 240 dynes. 26 
5) Pulmonary venous hypertension in clinically indicated level 3 CPET: rest mPAP 
>25 mmHg and rest PCWP >15 mmHg.27 
6) Exercise induced pulmonary arterial hypertension in clinically indicated level 3 
CPET: In patients > 50 years of age: peak mPAP > 33 mmHg and PVR > 168 
dynes; and in patients > 50 years of age: peak mPAP > 33 mmHg and PVR > 
168 dynes. 28 
7) Exercise induced pulmonary venous hypertension in clinically indicated level 3 
CPET: rest mPAP ≥ 25 mmHg and PCWP ≤ 15 mmHg and, in patients ≤ 50 years 
of age: PCWP > 19 mmHg and PVR < 108 WU; or in patients > 50 years of age 
peak PCWP > 17 mmHg and PVR ≤ 168 WU. 27 
8) Persistent hypotension during or after the clinically indicated level 3 CPET: SBP 
< 90 mmHg for more than 5 minutes. 
9) Refractory arrhythmia during or after the clinically indicated level 3 CPET. 
  
  
IV. Subject Enrollment 
  
Subjects will be recruited from the population of adults between 18 and 80 years old 
who are scheduled to undergo a clinically indicated level 3 CPET at Brigham and 
Women’s Hospital (BWH). These subjects are scheduled for this test for the evaluation 
of unexplained exertional intolerance and, for the most part, are seen in the Dyspnea 
Clinic at Brigham and Women’s Hospital prior to testing. This clinic is staffed by 
multiple pulmonary physicians besides the principal investigator. Dr. Systrom, the 
principal investigator, who is also the director of the Dyspnea Clinic and the Advanced 
CPET Program at BWH, will be the physician who will conduct the cardiopulmonary 
exercise tests for every patient. 
  


 
7 
Subjects will be pre-screened as described above. Based on the medical history 
obtained from the electronic medical record, potential subjects will be recruited. A 
recruitment letter signed by Dr. Systrom, as the physician who will perform the 
cardiopulmonary test for all eligible subjects, will preferably be mailed, or, if not 
possible,  emailed through a send secure e-mail to potential eligible subjects. 
  
Prospective subjects will be given a one-week period to contact the research staff at 
either their emails or their phone numbers to notify if they are or not interested in 
knowing more about the study. If after that period, the research staff has not heard 
from the patient, the prospective subject will be called by a research staff to see if they 
are interested. If a prospective subject is interested in knowing more about the study, 
a detailed explanation of the study protocol will be given and pre-screening questions 
will be asked to determine eligibility, if data was not clearly stated in their electronical 
medical records, (please see attached telephone script). If a subject meets the initial 
eligibility criteria, ample time will be provided to ask questions, and the study staff will 
reinforce the subject that participation is voluntary, that they do not have to participate, 
and that the decision not to participate will not affect their care, now or in the future. If 
the subject agrees, a consent form will be sent through a send secure e-mail or mailed 
to read. Study staff will encourage the subject to share the consent form with health 
care providers and/ or relatives. A follow up phone call or email will be held with the 
patient to assess if they would like to participate in the study at least 48 hours after 
they receive the consent form and before his/her scheduled iCPET. 
  
Subjects may be enrolled directly from Dr. Systrom’s Dyspnea Clinic visit at BWH. In 
this case, the research study will be mentioned in a clinic visit and, if the prospective 
subject expresses interest in the study, the consent form will be given to the 
prospective subject to read at home and research staff will call the prospective subject 
to go over the study, assess eligibility, answer questions, and see if the prospective 
subject would like to participate in the study. Prospective subjects will be encouraged 
to discuss this study with other healthcare providers to obtain additional guidance. 
  
Prospective subjects, who are not Dr. Systrom’s patients, but who communicate with 
any of the research team to express their interest to participate in the study, regardless 
of how they find out about the study, will be pre-screened and provided with the 
consent form if potentially eligible.   
  
Prospective subjects who are taking pyridostigmine would be asked to discuss holding 
their medication at least one week before the study with their prescribing physician. 
  
The day of their level 3 CPET, prospective subjects will be given another copy of the 
consent form and, once all questions are answered and if the prospective subjects still 
want to participate, they will sign the consent form. Study staff will then take a physical 
copy and an electronic copy of the consent form signed by either Dr. Systrom or a 
licensed co-investigator and the subject, give the physical copy of the signed consent 
form to the subject and add the original signed consent form to the subjects’ source 
documents and upload the electronic copy to their electronic medical records.  
  
All subjects will also be participating in the Pulmonary Vascular Disease Research & 
Clinical Care Tissue Bank (Protocol # 2011P000272). 
  


 
8 
  
V. Study Procedures 
  
Patients will undergo a clinically indicated level 3 CPET. The standard of care for this 
involves placing a pulmonary artery catheter/right heart catheter and a radial artery 
catheter in the cardiac catheterization lab. Patients are then transported to the 
exercise lab for a cardiopulmonary exercise test with both catheters in place. Usual 
practice involves obtaining hemodynamic measurements at rest, then during free-
wheeling (no resistance), then with increasing resistance until they reach peak 
exercise determined by heart rate response and gas exchange measurements. 
Hemodynamics are recorded every minute during exercise. Arterial and mixed venous 
blood samples are collected every minute during exercise. The CPET concludes when 
the patient asks to stop the exercise test or after peak exercise has been achieved. 
Hemodynamic measurements and blood draws also occur during the recovery period. 
  
The details of the study after the clinically indicated CPET are as follows: 
1. After a clinically indicated level 3 CPET, a subject’s hemodynamic waveforms 
and ventilatory variables will be reviewed for the final exclusion criteria. The 
clinically indicated level 3 CPET will be supervised by the physician 
investigators. 
2. The subjects who meet the final eligibility criteria and who attest that they can 
do the recovery cycling will be given a single dose of pyridostigmine 60 mg PO 
versus placebo. This will be administered by either the physician investigators, 
or a physician’s assistant. Both the patient and physician will be blinded to the 
study drug. Drug selection will be randomized. 
3. The subject will remain in the exercise lab to rest for 50 minutes. They will be 
under the supervision of a physician or physician’s assistant for this time-period 
and continuously monitored using a portable cardiac monitor. The pulmonary 
artery and radial artery catheters will be flushed with heparinized-saline by the 
exercise physiologists, physician investigator or physician’s assistant to ensure 
that the lines will remain patent, as it would be usually done in order to draw 
the 1-hour post lactate samples. 
4. After 50 minutes, the patient will perform the recovery cycling. Usual practice 
of the cardiopulmonary exercise test involves obtaining arterial and mixed 
venous blood samples every minute during exercise. During recovery cycling, 
arterial and mixed venous blood samples will only be obtained two times: during 
rest and at peak exercise. This adds 6 mL of blood drawn in addition to the 
blood drawn during the clinically indicated CPET. Recovery cycling will be 
performed under supervision of the physician investigators. 
5. Key differences between recovery cycling and the clinically indicated CPET 
include: 
• Pulmonary capillary wedge pressure and other hemodynamic 
parameter’s measurements taken only two times (rest and peak 
exercise), minimizing the already small risk of pulmonary artery rupture. 
• Elimination of a lactate level drawn one hour after exercise completion, 
expediting catheter removal. 
• Minimization of blood drawn to 6 mL, compared to 45 mL drawn during 
the clinically indicated CPET. 
  


 
9 
6. Additionally, a demographics questionnaire and the ME/CFS ICC 1-10 
Symptom Severity Questionnaire29 will be administered to all subjects. These 
will either be administered through REDCap or on paper and results stored in 
REDCap and in the subjects study chart. 
  
7. The modified Borg Scale30 for assessing dyspnea and fatigue will be 
administered after the clinically indicated CPET and after recovery cycling with 
the study drug. This will be used to assess subjective responses to the study 
drug. 
  
8. Subjects will receive a follow up phone call or email within the following week, 
ideally within two days after the study to assess if they experience any adverse 
events from the study. This will be documented for every patient and if severe 
adverse events happen, action will take place to improve subject’s health and 
the IRB will be notified. 
  
  
  
VI. Biostatistical Analysis 
  
Statistical analysis will be performed with either STATA, SAS, or Graphpad Prism. 
With 80% power to detect a 10% difference in oxygen uptake at peak exercise, we 
estimate the need to enroll 60 patients. This number also accounts for screening 
failures. 
  
 


 
10 
 
   
Y-axis: Sample size 
X-axis: Single-arm sample size 
Upper green line: 90% power 
Middle purple line: 80% power 
Lower teal line: 70% power 
  
VII. Risks and Discomforts 
  
There are risks of side effects from pyridostigmine administration, such as nausea, 
diarrhea, muscle cramping and twitching, excessive drooling, bradycardia, and 
excessive ocular tearing. 
  
Risks of blood draws include a small risk of infection, lightheadedness, and/or fainting 
because of the additional blood that is being drawn. 
  
Risks of the arterial catheter remaining in place for recovery cycling include pain, 
bleeding, swelling, or redness where the catheter is placed, brief loss of pulse due to 
blood flow problems in the artery, damage to the artery wall or nearby nerves, 
inadvertent removal, and infection (infection is rare and happens in less than 1 in 100 
cases). Placement of a radial arterial line in the wrist can cause decreased blood flow 
to the hand that may require surgery to correct. This is very rare and has not happened 
when the catheter is left in place for only a few hours for research purposes. 
  
There are risks related to the pulmonary arterial (PA) catheter remaining in place for 
the rest period and recovery cycling. Risks may include arrhythmias, tachycardia, 
bradycardia, and infection. This is minimized by careful observation for any of these 
events during the clinically indicated CPET.   
  
There is a risk of pulmonary artery injury during the PCWP measurements (balloon 
inflation). This is minimized by limiting this to three inflations during recovery cycling 
and knowing exactly how much air is needed to inflate the balloon based on the prior 
CPET. 
Among 
approximately 
7,000 
invasive 
CPETs 
performed 
between 


 
11 
Massachusetts General Hospital and Brigham and Women’s Hospital, there has only 
been one case of pulmonary artery rupture. 
  
While exercise testing is generally safe, risks related to recovery cycling include heart 
attack, stroke, and dangerous arrhythmias. The risks are minimized by requiring the 
successful and uneventful preceding, clinically indicated CPET. 
  
The risks listed above are identical to those during the clinically-indicated CPET. Both 
the administration of pyridostigmine or placebo and cardiopulmonary exercise testing 
will be performed under the direct supervision of the physician investigators. 
  
There is a risk that the subject’s private health information will be released to the 
public. All subjects participating will be assigned a specific study number.  All 
information will be de-identified. 
 
For those patients who are taking pyridostigmine and are asked to withhold it before 
the study, mild discomfort might arise. Although no significant withdrawal symptoms 
have been linked to pyridostigmine withholding, in some subjects some mild ME/CFS 
symptoms might return transiently. Subjects who are asked to withhold pyridostigmine 
before the study will be instructed to resume taking it as soon as the test is completed. 
 
VIII. Potential Benefits 
  
ME/CFS remains a prominent medical issue with very little known about its 
pathophysiology and treatment. At present, little is also known about the relationship 
between the autonomic nervous system and cardiopulmonary hemodynamics during 
exercise. Information obtained from this research will contribute to our understanding 
of normal human physiology and the pathophysiology behind ME/CFS. 
  
Subjects enrolled in this study may not benefit from this study. Research obtained from 
their participation may benefit other patients in the future who suffer from exertional 
intolerance.  
  
IX. Monitoring and Quality Assurance 
  
The principal investigators will perform this function. This study is an investigator-
initiated study and will be conducted only at Brigham and Women’s Hospital. The 
range of possible study events that could have an important impact on the risks and 
benefits of research participants is narrow. Continuous monitoring of events by the 
investigator will prompt reporting of serious adverse events to the IRB and, when 
applicable, the FDA, or others. 
  
All reports of adverse events, breaches of confidentiality, complaints, and 
unanticipated problems will be reviewed by the principal investigator and will be 
submitted to the IRB per the Partners HRC policy. 
  
The principal investigators will be required to guarantee that research staff prepare 
and maintain adequate and accurate case histories designed to record all 
observations and other data pertinent to the study for each study participant. All study 
data recorded will be documented in the database maintained by the exercise lab and 


 
12 
source documents. The research team will meet weekly to review the accuracy and 
completeness of the consent forms and data collected per protocol. 
  
To ensure privacy and confidentiality, codes will be substituted for identifiers and 
access to the data will be restricted to the principal investigator and the research team. 
The research staff will be reminded at regular intervals of the importance of 
maintaining research subject confidentiality. Research records are kept electronically 
in either a Partners encrypted laptop or desktop computer kept at the Thorn building 
and, whenever possible, physically in a locked cabinet at BWH, PBB-Clinics 3. No 
information on subjects and biological samples will be sent outside of the Partners 
network. Materials will not be maintained for use outside the scope of this protocol. 
  
   
  
  
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