Ketone Ester Effects on Biomarkers of Brain Metabolism and Cognitive Performance in Cognitively Intact Adults >= 55 Years Old. A Double-Blinded Randomized Controlled Clinical Trial.
Phase: NA
Conditions: Metabolic Syndrome, Normal Cognition
Sponsor: National Institute on Aging (NIA)

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Abbreviated Title: Oral Ketone Ester effects on brain function
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Abbreviated Title: Oral Ketone Ester effects on brain function
Protocol #: 20AG0087
Version Date: 10/29/2024
Title: Ketone ester effects on biomarkers of brain metabolism and cognitive performance in 
cognitively intact adults ≥ 55 years old. A double-blinded randomized controlled clinical trial 
Principal Investigator: Dimitrios Kapogiannis, MD
NIH Principal Investigator:
Laboratory of Clinical Investigation
National Institute on Aging
NIA Clinical Unit at Medstar Harbor Hospital
3001 S. Hanover St, NM531
Baltimore, MD 21225
Phone: 410-350-3953
E-mail: kapogiannisd@mail.nih.gov
Investigational Agents (if applicable):
Drug Name:
IND Number:
N/A
Sponsor:
N/A
Manufacturer:

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TABLE OF CONTENTS 
TABLE OF CONTENTS ................................................................................................................2
STATEMENT OF COMPLIANCE ................................................................................................6
1
PROTOCOL SUMMARY.......................................................................................................7
1.1
Synopsis ............................................................................................................................7
1.2
Schema..............................................................................................................................8
1.3
Schedule of Activities (SOA) .........................................................................................10
2
INTRODUCTION .................................................................................................................14
2.1
Study Rationale...............................................................................................................14
2.2
Background .....................................................................................................................14
2.3
Risk/Benefit Assessment.................................................................................................20
2.3.1
Known Potential Risks ............................................................................................20
2.3.2
Known Potential Benefits........................................................................................22
2.3.3
Assessment of Potential Risks and Benefits............................................................23
3
OBJECTIVES AND ENDPOINTS.......................................................................................24
4
STUDY DESIGN ..................................................................................................................28
4.1
Overall Design ................................................................................................................28
4.2
Scientific Rationale for Study Design.............................................................................29
4.3
Justification for Dose ......................................................................................................29
5
STUDY POPULATION........................................................................................................30
5.1
Inclusion Criteria.............................................................................................................30
5.2
Exclusion Criteria ...........................................................................................................31
5.3
Inclusion of Vulnerable Participants...............................................................................32
5.4
Lifestyle Considerations .................................................................................................33
5.5
Screen Failures................................................................................................................33
5.6
Strategies for recruitment and retention..........................................................................33
5.6.1
Costs ........................................................................................................................34
5.6.2
Compensation ..........................................................................................................34
6
STUDY INTERVENTION ...................................................................................................35
6.1
Study Interventions(s) Administration............................................................................35
6.1.1
Study Intervention Description................................................................................35
6.1.2
Dosing and Administration......................................................................................35

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6.2
Preparation/Handling/Storage/Accountability................................................................36
6.2.1
Acquisition and Accountability ...............................................................................36
6.2.2
Formulation, Appearance, Packaging, and Labeling...............................................36
6.2.3
Product Storage and Stability ..................................................................................36
6.3
Measures to Minimize Bias: Randomization and Blinding ............................................37
6.4
Study Intervention Compliance ......................................................................................38
6.5
Concomitant Therapy......................................................................................................38
7
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL .....................................................................................38
7.1
Discontinuation of Study Intervention............................................................................38
7.2
Participant Discontinuation/Withdrawal from the Study................................................39
7.3
Lost to Follow-up............................................................................................................39
8
STUDY ASSESSMENTS AND PROCEDURES.................................................................40
8.1
Screening Procedures......................................................................................................40
8.2
Efficacy Assessments......................................................................................................40
8.2.1
Clinical Evaluations.................................................................................................40
8.2.2
Neuropsychological Assessment .............................................................................40
8.2.3
Magnetic Resonance Spectroscopy (MRS)/Magnetic Resonance Imaging (MRI):41
8.2.4
Compliance Assessments.........................................................................................42
8.2.5
Biospecimen Evaluations.........................................................................................43
8.2.6
Correlative Studies for Research .............................................................................45
8.2.7
Samples for Genetic/Genomic Analysis..................................................................47
8.3
Safety and Other Assessments ........................................................................................48
8.4
Adverse Events and Serious Adverse Events .................................................................49
8.4.1
Definition of Adverse Event....................................................................................49
8.4.2
Definition of Serious Adverse Events (SAE) ..........................................................49
8.4.3
Classification of an Adverse Event..........................................................................49
8.4.4
Time Period and Frequency for Event Assessment and Follow-Up........................51
8.4.5
Adverse Event Reporting.........................................................................................51
8.4.6
Serious Adverse Event Reporting............................................................................51
8.4.7
Reporting of Pregnancy ...........................................................................................52
8.5
Unanticipated Problems ..................................................................................................52

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8.5.1
Definition of Unanticipated Problems (UP) ............................................................52
8.5.2
Unanticipated Problem Reporting ...........................................................................52
9
STATISTICAL CONSIDERATIONS ..................................................................................52
9.1
Statistical Hypothesis......................................................................................................52
9.2
Sample Size Determination.............................................................................................53
9.3
Populations for Analyses ................................................................................................53
9.3.1
Evaluable for toxicity ..............................................................................................53
9.3.2
Evaluable for objective response.............................................................................53
9.4
Statistical Analyses .........................................................................................................54
9.4.1
General Approach....................................................................................................54
9.4.2
Analysis of the Primary Endpoints..........................................................................54
9.4.3
Analysis of the Secondary Endpoint(s) ...................................................................54
9.4.4
Safety Analyses .......................................................................................................54
9.4.5
Baseline Descriptive Statistics.................................................................................55
9.4.6
Planned Interim Analysis.........................................................................................55
9.4.7
Sub-Group Analyses................................................................................................55
9.4.8
Tabulation of individual Participant Data................................................................55
9.4.9
Exploratory Analyses...............................................................................................55
10
REGULATORY AND OPERATIONAL CONSIDERATIONS.......................................55
10.1
Informed Consent Process...........................................................................................55
10.1.1
Consent/Assent Procedures and Documentation.....................................................55
10.1.2
Participation of Subjects who are/become Decisionally Impaired..........................56
10.2
Study Discontinuation and Closure.............................................................................56
10.3
Confidentiality and Privacy.........................................................................................56
10.4
Future use of Stored Specimens and Data...................................................................57
10.5
Safety Oversight..........................................................................................................58
10.6
Clinical Monitoring .....................................................................................................58
10.7
Quality Assurance and Quality Control ......................................................................58
10.8
Data Handling and Record Keeping............................................................................59
10.8.1
Data Collection and Management Responsibilities.................................................59
10.9
Protocol Deviations .....................................................................................................59
10.9.1
NIH Definition of Protocol Deviation .....................................................................59

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10.10
Publication and Data Sharing Policy...........................................................................59
10.10.1 Human Data Sharing Plan .......................................................................................59
10.11
Conflict of Interest Policy ...........................................................................................60
11
ABBREVIATIONS ............................................................................................................60
12
REFERENCES ...................................................................................................................61

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STATEMENT OF COMPLIANCE
The trial will be carried out in accordance with International Conference on Harmonization Good 
Clinical Practice (ICH GCP) and the following: 
• United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 
CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812) 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training.
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study.  In addition, all changes to the consent form will be IRB-approved; a 
determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form.

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1
PROTOCOL SUMMARY
1.1
SYNOPSIS
Title:
Ketone ester effects on biomarkers of brain metabolism and cognitive  
performance in cognitively intact adults ≥ 55 years old. A double-
blinded randomized controlled clinical trial. 
Study 
Description:
We hypothesize that supplementation with a ketone ester drink [Ketone 
Ester (KE)] compared to placebo, in cognitively intact adults ≥ 55 years 
old with Metabolic Syndrome (MetS), will (i) increase peripheral and 
brain ketone levels [primarily β-hydroxybutyrate (BHB) and secondarily 
acetoacetate (AcAc)], (ii) improve neuronal/astrocytic insulin resistance 
(IR) and induce a change in neuronal/astrocytic metabolism as reflected 
on blood Extracellular Vesicle (EV) biomarkers, (iii) improve cognitive 
performance, (iv) boost mitochondrial function in muscle, and (v) 
change gut microbiome. These effects will be examined acutely, after 
single-dose administration, and chronically, after 28 days on the 
supplement x 3 times per day. The changes in EV biomarkers and 
cognition will be associated with the elevation of ketones in brain. The 
study will involve a Screening Visit and three additional Visits to assess 
acute effects, compliance and chronic effects, respectively, and a follow-
up visit to obtain DNA.
 
Objectives:
Primary: To investigate the change in brain concentration BHB, using 
brain Magnetic Resonance Spectroscopy, after 28 days of 
supplementation with the KE, compared to baseline and placebo.
Secondary: To test the hypothesis that genetic factors may affect the 
response to the KE supplement. 
Endpoints:
Primary: To detect with brain MRS, a significant change in the 
concentration of BHB, after 28 days of supplementation with the KE 
compared to baseline and placebo
Secondary: To assess whether genetic factors modulate the response to 
the KE supplement. 
Study 
Population:
Males or females, of age ≥ 55 years who meet the criteria for MetS and 
are cognitively intact. 
Phase:
N/A – study of a dietary supplement.

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Description of 
Sites/Facilities 
Enrolling 
Participants:
The study will take place at a single site, at the NIA Clinical Unit at the 
Medstar Harbor Hospital, Baltimore 
Description of 
Study 
Intervention:
Oral KE drink [containing 25 g KE/(R)-3-hydroxybutyl (R)-3-
hydroxybutyrate] vs isocaloric Placebo drink (containing dextrose) x 3 
times/day x 28 ± 3 days 
Study Duration:
25 months
Participant 
Duration:
Up to 59 days (Screening Visit is followed by an initiation visit within 28 
days and then 28 ± 3 days receiving the drink supplement). One additional 
follow-up visit to draw blood for DNA collection.
1.2
SCHEMA
Pre-Visit phone call/
Visit 1 
Screening Visit
Visit 2
Day 1 on
supplement
N: 150 for screening ceiling; N: 50 for complete data.
  Pre-Visit phone call: review inclusion/exclusion criteria. 
Visit 1 (Screening): informed consent. screen by inclusion and exclusion 
criteria; medical history and physical exam; review of concomitant 
medications; demographics; vital signs and anthropometric measurements 
(height, weight, waist circumference); blood draws for clinical labs; 
cognitive assessment for eligibility (CDR, MOCA); urine test for 
pregnancy; meals/snacks.
Baseline assessments: blood draw for clinical labs and EV biomarkers; cognitive testing; 
MRI/MRS; stool samples for gut microbiome, urine pregnancy test, optional thigh MRS
First dose
Acute effects: blood draw (glucose, BHB, HbA1C, insulin, EV biomarkers); MRI/MRS
Urine collection
Arm 2 
Placebo
 25 participants
Arm 1 
Ketone Ester 
25 participants
Randomize

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Visit 3
Day 14 ± 3 on
supplement 
Visit 4
Day 28 ± 3 on
supplement
Note: During the 28 ± 3-day period, participants will self-administer the intervention at home, 
three times per day. On Visits 2, Visit 3 and Visit 4, the first dose of the daily intervention will 
be given at the NIA Clinical Unit, where the Visits will take place.
Visit 5 (Follow-up visit)
Informed consent and blood draw for DNA collection. 
Compliance assessment. Dose.
 Blood draw for clinical labs and EV biomarkers, urine pregnancy test, 
Credibility/Expectancy Questionnaire
Last dose. Chronic effects 
assessments: urine pregnancy test, 
blood draw for clinical labs (glucose, 
BHB, HbA1C, insulin, EV 
biomarkers) before and after last dose; 
MRI/MRS before and after last dose; 
optional thigh MRS; cognitive testing 
after last dose; Credibility/Expectancy 
Questionnaire; urine collection; stool 
samples for gut microbiome.

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1.3
SCHEDULE OF ACTIVITIES (SOA)
 
Procedure
Pre-
Visit 
phone 
Screen
Visit 1
Visit 2
Visit 3
Visit 4
Visit 5
Screening
Baseline 
assessments – 
first dose – acute 
effects 
assessments 
Compliance 
Assessment 
Last dose. 
Chronic effects 
assessments
Timing
Within 28 days of 
V1
Day 1 on 
supplement
Day 14 (+/-3 days) 
on supplement
Day 28 (+/-3 
days) on 
supplement
Informed Consent
X
X
Screening of Inclusion/Exclusion 
Criteria
X
X
Demographics
X
Medical History
X
Review of concomitant medications
X
X
X
X

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Physical Exam
X
X
Vital signs 
X
X
X
X
Nursing assessment
X
X
X
X
Anthropometric measures [Height 
(cm), Weight (kg), waist 
circumference (cm or in)] *Height to 
be measured during V1 only. Weight 
and waist circumference to be 
measured during V1 and V4.
X
X
Blood draw for screening clinical labs: 
See section 8.2.5 
X 
(fasting)
Blood draw for clinical labs and EV 
biomarkers. See section 8.2.5
X (fasting/before 
supplement)
X (fasting/before 
supplement)
X 
(fasting/before 
supplement)
Blood draw for DNA collection (for 
genetic studies): See section 8.2.5
X
Serum β-hydroxybutyrate
X (fasting and 
~60 min after 
supplement)
X (fasting and ~60 
min after 
supplement)
X (fasting and 
~60 min after 
supplement)
Stool samples
X (within 24 h 
before the visit)
X (within 24 h 
before the visit)
Urine BHB 
X (collected 
during the visit)
X (collected 
during the visit)

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Urine drug screen
X
 
Urine Pregnancy test 
X
X
X
X
Cognitive assessment for eligibility 
(CDR, MOCA)
X
Randomization (after eligibility 
determined) by NIA Pharmacist
X
Ketone Ester or Placebo dispensed by 
NIA pharmacist
X (first of three 
daily doses 
received in-
house)
X (first of three 
daily doses received 
in-house)
X first of three 
daily doses 
received in-
house)
Self-administration of Ketone Ester or 
Placebo x 3 times per day
Throughout the 28-day period 
MRI/MRS Brain
X (before and 
~75 min after 
supplement)
X (before and 
~75 min after 
supplement)
Optional Thigh MRS
X (before after 
supplement)
X (before and 
after 
supplement)
Outcome cognitive testing
X (before the 
supplement)
X (~ 2.5 h after 
the supplement)
Review compliance logs of drinks 
X
X
Modified Credibility/Expectancy 
Questionnaire
X
X

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Meals/Snacks
X
X
X
X
X
Phone calls to monitor compliance/ 
elicit AEs
Once between Visits 2, 3 and once between Visits 3, 4)
Adverse Event monitoring
Throughout the 28-day period (4 Visits and weekly phone calls)

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2
 INTRODUCTION
2.1
STUDY RATIONALE
Currently, there is no medication to treat or halt the progression of Alzheimer’s disease (AD). 
According to the standard of care, the only drug categories prescribed in AD are cholinesterase 
inhibitors and memantine; both drugs have symptomatic but not disease-modifying effects. 
Ongoing clinical trials are currently testing new drugs directed against a variety of different targets 
1. Around a third of drugs that are being tested in phase 2/3 trials target neuropsychiatric symptoms 
or aim at symptomatic relief 1. Of the remaining two thirds, most target the beta-amyloid (A) 
and/or tau pathologies or have  different actions such as neuroprotection, decrease of inflammation 
or improvement of metabolism 1. Of the very few drugs that aim to improve metabolic function 1, 
no drug is targeted towards providing an alternative to glucose as an energy source to the brain, 
despite the abundance of evidence implicating brain glucose hypometabolism as a core feature in 
AD 2-5.  
The idea of providing an alternate energy fuel, such as ketone bodies, either endogenously through 
exercise or diet, or exogenously through dietary supplements, to overcome brain glucose 
hypometabolism in AD is not new 5-10. Administration of ketogenic Medium Chain Triglycerides 
(MCTs) as a dietary supplement has already been tested with promising but inconclusive results 
in terms of cognitive improvement in AD patients 11. Direct supplementation of ketones in the 
form of ketone ester (KE) or salts (KS), has never been systematically tested in AD. Currently, a 
popular exogenous ketone in the form of a ketone ester (KE) is (R)-3-hydroxybutyl (R)-3-
hydroxybutyrate. [Note: in this protocol we will refer to the (R)-3-hydroxybutyl (R)-3-
hydroxybutyrate simply as Ketone Ester (KE) and to the oral drink formula that consists mainly 
of KE and water as  KE drink]. Ketone Ester (KE) is known to be the most potent form of 
exogenous ketone, and has been tested in wild type and AD model rodents with positive 
behavioral/cognitive and disease pathology outcomes 12-14. In our view, a significant evidence gap 
exists between demonstrated disease pathology-modifying and pro-cognitive effects of the KE in 
animals and the lack of evidence for any pro-cognitive effects in healthy or diseased humans. 
Before contemplating a clinical trial of a KE drink in patients with AD, further proof of concept 
for target engagement and pro-cognitive effects in humans is required. The goal of this clinical 
study is to bridge this gap by testing if a KE drink can increase brain ketone levels (demonstrating 
target engagement and a pharmacodynamic effect), modulate neuronal metabolism and AD-related 
cascades [as reflected on plasma Extracellular Vesicle (EV) biomarkers] and improve cognitive 
performance. The study will be conducted in a cognitively intact population characterized by brain 
hypometabolism in a similar regional pattern as the one observed in AD, albeit to a lesser degree 
15,16. Should results be positive in a population with AD-like brain hypometabolism, a similar study 
involving individuals with AD may be contemplated in the future.  
2.2
BACKGROUND 
2.2.1 Evidence from animal studies reveals the potential of Ketone Ester (KE) for improving 
cognition and pathology in AD
In a study involving healthy rats that consumed KE vs isocaloric placebo for five days as part of 
their diet (30% of calories), the KE group made more correct decisions before making a mistake 

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on a 8-arm radial maze test, suggesting improved cognition 13. Chronic KE administration was 
compared with an isocaloric placebo in a triple-transgenic AD (3xTgAD) mouse model study, 
revealing that KE improves learning and spatial memory (figure 1) while reducing Aβ and 
hyperphosphorylated tau deposition in the hippocampus, amygdala and cortex (figure 2) 14. In 
another study, KE administration resulted in increased hippocampal d-β-hydroxybutyrate, 
decreased oxidative status, and greater energy of ATP hydrolysis in in the 3xTgAD mouse model 
12. On the other hand, low-carbohydrate/high-fat ketogenic diets did not show any benefit in terms 
of improving cognition and reducing AD pathologies in AD mouse models 17,18, which points 
towards the superiority of KE compared with ketogenic diets as a ketogenic intervention in AD.
Figure 1. A Ketone Ester Diet (43.5% carbs, 24% protein, 8.2% fat and 21.5% Ketone ester) improved 
performance in hippocampus-dependent water maze tests of spatial memory, of 3xTgAD mice at 12 and 
15 months on the diet (phase 1 and 2, respectively). CHO: Carbohydrate-enriched diet; KET: Ketone 
Ester diet 14.

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Figure 2. A Ketone Ester Diet (43.5% carbs, 24% protein, 8.2% fat and 21.5% Ketone ester) 
decreases counts of Aβ and pTau immunoreactive cells in multiple regions of 3xTgAD mice 
brains. CHO: Carbohydrate-enriched diet; KET: Ketone Ester diet 14.
2.2.2 Human brain imaging studies suggest that ketones could compensate for the brain 
glucose hypometabolism observed in AD and other conditions with AD-like hypometabolism
Observational neuroimaging studies using fluoro-deoxy-glucose positron emission tomography 
(FDG-PET) and Magnetic Resonance Spectroscopy (MRS) have revealed that patients with Mild 
Cognitive Impairment (MCI) or AD 3, cognitively normal individuals with family history of AD 
or genetic risk for AD, and cognitively normal individuals of older age show a similar regional-
specific pattern of brain glucose hypometabolism (typically, hypometabolism predominates in 
lateral parietal-temporal and medial parietal/precuneal regions early in the disease, before 
extending into frontal and occipital regions) 2,4,15. Interestingly, cognitively normal individuals 
with insulin resistance (IR) also show Alzheimer’s-like reductions in cerebral glucose metabolism 
in association with their degree of peripheral insulin resistance (IR) (e.g. correlating with 
Homeostatic Model Assessment of IR (HOMA-IR)) 15,16. On the other hand, PET scan studies 
using the ketone tracer [11C]-acetoacetate ([11C]-AcAc), have found that metabolism of 
Acetoacetate (AcAc) in regions characterized by glucose hypometabolism is normal in individuals 
with MCI/AD 4,19. This observation supports the hypothesis that an increase of ketones availability 
in brain could potentially compensate for the energetic deficit that is present in AD and other 
conditions, such as IR 5,15 .
2.2.3 Interventions that induce mild peripheral ketosis are associated with modestly positive 
cognitive outcomes in individuals with AD

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Most clinical studies involving ketogenic interventions as a means of treatment for AD have been 
conducted using oral Medium Chain Triglycerides (MCTs) supplements 6,7,11,20-22. We recently 
published a meta-analysis of human studies on that topic, showing that supplementation with oral 
MCTs induced mild peripheral ketosis (β-hydroxybutyrate (BHB) ~ 0.5mM) and marginally 
improved a composite measure of global cognition 11. We hypothesize that the modest and 
somewhat inconsistent effects of this ketogenic intervention on cognition is due to the mild levels 
of ketosis induced. Thus, an intervention that would increase peripheral ketones at greater levels 
could be associated with more pronounced and consistent cognitive benefits.
2.2.4 Oral Ketone Ester (KE) intake safely induces robust peripheral ketosis in humans 
Supplementation with KE has been shown to produce peripheral ketosis acutely at convenient, 
tolerable and safe doses 23-26, much more robustly compared with other ketogenic interventions 
(up to levels of BHB ~ 4.0 mM). The level of ketosis achieved with a single KE dose is 
impressively comparable to ketosis produced after 2 weeks starvation (4.0 – 7.0 mM) 27. In 
comparison, peripheral BHB levels barely reach 1 mM after 2 weeks of low carbohydrate-diet/high 
fat diets 28,29  . Medium chain Triglycerides supplementation which is considered the second most 
potent to KE ketogenic intervention, barely induces peripheral BHB of 0.5 mM after a single 30-
g dose 30. Importantly, dosing with 30 g of MCTs is associated with very frequently reported (~40-
50 %) GI side effects, such as diarrhea and abdominal discomfort 31 (see figure 3 for a comparison 
between ketosis induced by the two most potent ketogenic supplements, MCTs and KE).  
Figure 3. Left: Relationship between dose of MCTs and plasma BHB. Data were collated by dose 
from several human studies. 30Note: 40-50% of subjects experience either abdominal discomfort 
or diarrhea after a single 30 g dose of MCTs 31Right: Blood BHB kinetics following isocaloric KE 
and DEXT drinks in 15 subjects at rest. Values are means ± SEM. *P < 0.05 difference between 
KE and DEXT. This BHB elevation is induced by a dose that corresponds to 27.7 gr in a 70-kg 
person. 32Note: This dose of KE has been associated with mild nausea in only 0.3% of drinks 
administered in a 28-day study 33
2.2.5 Robust peripheral ketosis with a KE drink administration is likely associated with 
analogous availability of ketones in brain
Collective evidence from various studies shows that for a range of peripheral BHB between 0.02 
and 12 mM induced by several different ways and at physiological or pathological states (including 
AD), the brain’s ketone metabolism is proportional to the peripheral levels 15 (see figure 4). This 

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implies that the ability of a ketogenic intervention to provide ketones to the brain is directly 
analogous to its ability to raise ketone levels in the periphery. The cerebral metabolic rate (CMR) 
of ketone bodies (AcAc and BHB) can be quantified with the use of PET scans using ketone 
tracers, whereas their concentration in the brain tissue in vivo may be quantified by Magnetic 
Resonance Spectroscopy (MRS) 4,19,34. Although a head to head comparison of brain ketone 
metabolism assessed by PET and brain ketone concentration by MRS quantification has not been 
reported in the literature, their association is likely given that they both correlate closely with 
peripheral levels. Therefore, pharmacodynamic effects of oral KE supplementation could be 
assessed in a study, in which peripheral ketone levels are measured in plasma and brain ketone 
levels are measured by MRS. The proposed study intends to measure both and examine their 
associations with biomarkers of neuronal metabolism and cognition. 
Figure 4. Data collated from multiple studies reveal the direct, linear relation between plasma 
ketone concentration and brain ketone uptake in: AD and healthy age-matched controls (beige 
and orange circles), postprandial state (yellow boxes), AD and healthy adults from a second study 
(light and dark green triangles), AD and healthy adults from a third study (light and dark blue 
rhombuses), BHB infusion (gray triangles), 40-day fast (black triangles), 60-day fast (white box); 
CMR: Cerebral Metabolic Rate; Two relationships are shown, one for plasma BHB vs rate of 
brain BHB uptake (solid line, R2 = 0.97; Y = 1.57X – 0.20; P < 0.0001) and one for plasma AcAc 
vs rate of brain AcAc uptake (dotted line, R2 = 0.83; Y = 3.46X – 0.03; P < 0.0001)30 
2.2.6 Induction of peripheral ketosis may be modulated by genetic factors
In a recently conducted randomized clinical trial of 5:2 Intermittent Fasting (IF) versus Healthy 
Living diet in older individuals with insulin resistance, we found that levels of BHB and AcAc in 
fasting blood increased across groups after 8 weeks of diet implementation.35 Interestingly, in 
exploratory analyses for genetic polymorphisms, the diets showed differential effects on induction 

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of peripheral ketosis. For SLC16A7 rs11173201 polymorphism, elevated ketogenesis with an 
increase in BHB and AcAc was only observed in individuals with A/A, but not A/G, genotype. 
Similarly, for APOE ε4 carrier status, AcAc only increased among ε4 carriers (see Figure 5). This 
implies that the ability of a ketogenic intervention to increase ketones in the periphery, and by 
extension to the brain, may be modulated by certain polymorphisms of genes that encode for 
proteins associated with ketone transport (i.e., MCT2) and overall brain metabolism (i.e., ApoE). 
The proposed study intends to explore how polymorphisms for these genes, polymorphisms for 
additional MCT coding genes, and additional genetic factors may affect the induction of peripheral 
and brain ketosis and additional outcomes.
 
Figure 5. Modulation of diet effects by SLC16A7 rs11173201 and APOE ε4 polymorphisms. 
Elevated ketogenesis with an increase in BHB was only noted in A/A individuals (Ftime*rs11173201 = 
4.63, P= 0.04; Ftime*rs11173201 A/A = 7.05, P = 0.01; Ftime*rs11173201 A/G = 0.25, P = 0.62). For serum 
AcAc, although no significant time*APOE ε4 interaction was noted (Ftime*ApoE = 0.11, P = 0.74), 
within genotype groups, increase was only observed among ε4 carriers (Ftime*ApoE ε4+ = 7.50, P = 
0.08; Ftime*ApoE ε4- = 3.23, P = 0.08).
2.2.7 Bridging the translational gap 
Despite promising evidence that KE improves cognition/disease pathology in animal models of 
AD and that less robust ketogenic interventions such as MCTs may improve cognition in humans 
with AD, there have been no human studies conducted on the pro-cognitive effects of a KE drink, 
in AD or in any other condition with AD-like brain hypometabolism. Importantly, existing 
evidence from imaging studies in humans supports that a KE drink is likely to robustly elevate 
ketones in brain where they can exert their potential positive actions. Our study aims to bridge the 
translational gap between findings in AD animal models and the absence of evidence for any 
effects on neuronal metabolism, AD-related cascades, and cognition, by studying the brain effects 
of a KE drink in a population that is characterized by AD-like brain glucose hypometabolism. The 
results and insights from this study are interesting for examining the impact of ketones in a highly 
prevalent state (Metabolic Syndrome (MetS)) characterized by (i) systemic and (ii) a degree of 

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brain IR and perhaps brain glucose hypometabolism. This may further motivate and help in the 
design of a future clinical trial implementing this supplement in AD.
2.3
RISK/BENEFIT ASSESSMENT
2.3.1
Known Potential Risks
Ketone Ester drinks: To our knowledge, which is based on the literature on KE and our 
communication with KE drink suppliers, there are no known serious reported or potential risks 
resulting from the use of this compound 10,25,26,32,36,37. Ketone Ester is already being sold in the 
market as a ketogenic supplement and is especially popular among athletes, such as cyclists. The 
main ingredient of KE drink that will be used in this study [(R)-3-hydroxybutyl (R)-3-
hydroxybutyrate)] is regulated as GRAS (Generally Recognized as Safe) substance by the FDA 
(https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=515). 
More 
relevantly, in a recent study in which participants were given 25 g of KE x 3 times per day for 28 
days (identical to the dose and duration we intend to study), there were no serious side effects 
resulting from the supplement 25. Mild nausea was the most commonly reported symptom but it 
was very rare (in 6 out of 2,106 drinks) 25. Other studies on KE had reported more frequent GI 
disturbances, but those had been attributed to higher doses used and/or co-administration of large 
quantities of milk with the supplement 36,38. The authors of one study reported that GI side effects 
post-KE administration might potentially be related to the bitterness of the KE compound 38. 
Finally, according to KE drink suppliers, there are currently no known reported adverse 
interactions between any drugs and the KE. To the best of our knowledge there is no other 
immediate or chronic physical, reproductive, psychological, social, legal, economic or other risk 
associated with the use of this supplement. Also, the only additional risks associated with the 
study’s procedures are those related to blood draws and MRI/MRS, which are all considered 
minimal.  
Below, we present the risks associated with the assessments conducted in this study:
•
Medical history and physical exam and nursing assessment: There are no significant 
risks to providing medical history and having a physical exam and nursing assessment. 
However, some psychological discomfort might result, if we find any abnormalities with 
potential clinical significance. Moreover, participants may be advised to have follow up 
with their own physicians, which may incur further inconvenience and costs. 
•
Anthropometric measurements: Some psychological discomfort might result from these 
procedures, if we find abnormal values (e.g. high blood pressure, abnormal weight).
•
Blood draws: There is a slight risk of pain, discomfort and bruising around the site where 
blood is drawn; bleeding and infection are rare side effects. To minimize this risk, the 
routine blood-drawing protocol will be followed, and pressure will be applied to the site. 
Some people experience feelings of light-headedness or dizziness after having blood 
drawn. This is may occur in up to 5% of participants. There is a remote risk of fainting and 
infection after having blood drawn. To reduce the risk of falling, we will monitor the 
subject closely and ask about these symptoms before we allow them to stand. Clean aseptic 
technique will be used by experienced staff while drawing your blood. The samples 
collected will be used to monitor the subject’s medical condition and for research purposes.

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•
Stored Samples: The greatest risk from the use of stored samples is the unplanned release 
of information from medical records. The chance that this information will be given to an 
unauthorized person without the participant’s permission is very small. We will not enter 
any experimental information (research results) into any other medical record and we will 
not release information to a third party unless specifically authorized by the participant.  
Possible problems with the unplanned release of information include discrimination when 
applying for insurance and employment. Some problems may occur if a participant 
discloses information or he or she agrees to have his or her medical records released.
•
Hepatitis Virus B, hepatitis virus C, and human immunodeficiency virus (HIV) 
Testing: This study requires the subject’s blood be tested for blood-transmitted diseases 
such as hepatitis virus B, hepatitis virus C, and human immunodeficiency virus (HIV). If 
the participant is infected with HIV, hepatitis B (positive HBsAG) or C (HCV RNA 
quantitative is detectable), he/she will not be able to participate in this study. We will tell 
the subject what the results mean, how to find care, how to avoid infecting others, how we 
report these infections, and the importance of informing any partners of possible risk.
•
Stool and urine samples: There are no known risks to providing stool or urine samples.
•
Urine drug screen: A urine toxicology screen will be performed during the screening visit. 
If the drug test is positive the results of the drug testing will be noted in participants’ 
medical record. Participants with a positive urine drug screen (and no prescription 
medication accounting for the positive test) will not able to participate in the study. 
Participants who do not want this information in their medical records should not 
participate in the study. The medical records can only be released with written agreement 
by the participant.  However, insurance companies may require individuals to release these 
records and may not provide insurance if they refuse. 
•
Urine pregnancy test: There is minimal risk to giving a urine specimen for pregnancy 
testing. Participants will not be able to participate in the study if the pregnancy test is 
positive. 
•
Questionnaires: There are no risks associated with completing questionnaires, but it may 
be time-consuming and inconvenient. 
•
Compliance Logs: There are no risks associated with completing compliance logs, but it 
may be time-consuming and inconvenient.
•
Cognitive test (also known as Neuropsychological Testing): Occasionally, cognitive 
tests may be tiring or stressful. Psychological testing may cause some people to feel 
anxious.
•
Modified Credibility/Expectancy Questionnaire: There are no risks associated with 
completing this questionnaire, but it may be time-consuming and inconvenient.  
•
Brain MRI/MRS and optional thigh MRI/MRS: People are at risk for injury from the 
MRI magnet if they have pacemakers or other implanted electrical devices, brain 
stimulators, dental implants, aneurysm clips (metal clips on the wall of a large artery), 
metallic prostheses (including metal pins and rods, heart valves, and cochlear implants), 
permanent eyeliner, implanted delivery pump, or shrapnel fragments. Welders and metal 
workers are also at risk for injury because of possible small metal fragments in the eye of 
which they may be unaware. Participants will be asked to complete an MRI screening form 
for each MRI. If any participant is found not to be eligible for the MRI or refuse the MRI, 
they will be disqualified from the study. In addition, all magnetic objects (for example, 

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watches, coins, jewelry, and credit cards) will be removed before entering the MRI scan 
room. Participants with history of surgeries, implanted devices, etc. will be asked to 
provide us with surgical reports mentioning the type of the clips, prostheses, etc. used. 
Every effort will be made to establish the MR compatibility or incompatibility of these 
implants, according to the safety standards of the NIA 3T MRI center, including obtaining 
reports by manufacturing companies for MR compatibility. The research staff will evaluate 
each person with a less severe history of claustrophobia on a case-by-case basis.
Regarding minor discomforts, rapidly changing magnetic fields of gradient coils, which 
are frequently used in research, can induce electrical fields in human tissue. This can result 
in stimulation of peripheral nerves and on rare occasions the participant may experience a 
mild sensation such as a light vibration, muscle twitching or poking of the skin with the 
effects varying from person to person. It has no lasting effects on the nervous system and 
the test will be stopped when the participant reports the symptoms to the MRI technician. 
The loud sounds (> 120 dB) emitted by the MRI machine during scanning require earplugs 
but may cause mild acoustic discomfort. 
Incidental findings on MRI that are non-life threatening and do not requiring further 
medically indicated diagnostic testing or treatment may occur in up to 5% of the 
participants.
Exercise Protocol for thigh muscle 31P MRS (Optional): There is a risk that an exercise 
procedure may cause fatigue, muscle cramping and/or strain. Participants will be asked to report 
any pain or discomfort as they complete the procedure(s) and based on the participant’s response, 
the procedure may be modified or terminated by the researcher.
•
Genetic testing (genotyping for APOE and other modulatory genes; DNA 
methylation): The DNA analysis that will be done as part of this study is for research 
purposes only. 
The primary risk associated with genetic testing in this study relates to the potential for a 
breach of confidentiality. In the event of a breach, genetic information could be misused, 
leaning to issues such as discrimination related to employment and/or life and/or health 
insurance. While such incidents are rare, they are possible. Additionally, access to genetic 
information could cause psychological distress or tension within families if a breach were 
to occur. 
Although there can be no absolute guarantees, every reasonable effort will be made to keep 
personally identifiable information confidential to prevent misuse and all results of the 
genetic testing are kept in a locked and secure manner at NIH. Participants are advised that 
their genetic information will not be revealed to others, including relatives, physicians, 
employers or insurance companies. Similarly, they will not receive information about other 
family members, nor will they receive any laboratory results from this type of testing. The 
privacy of the research information generated from this study is protected and considered 
highly confidential.
2.3.2
Known Potential Benefits
In general, individuals may have some transient benefits, such as increased subjective sensation of 
energy, mood and cognitive function.

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Single KE drink dose studies have shown improved glycemic response to OGTT 39 and decreased 
ghrelin and hunger 32; these effects which might be beneficial for our study population which 
consists of individuals with MetS. However, those benefits might not be clinically important since, 
in a 28-day study on the KE, there was no change in weight and glucose parameters 25. 
In healthy rats, a KE-diet improved their physical activity when compared with carbohydrate or 
Western type of diet 13. In high-performance athletes, co-ingestion of KE with carbohydrates 
significantly increased their cycling performance compared with carbohydrates only 37. Those 
results indicate that KE might be beneficial for the endurance level of any human and especially 
of those who exercise. 
As with physical activity, healthy rats showed improved cognition while on the KE supplement 13. 
In AD animal models, KE administration resulted in cognitive improvement, anxiety reduction 
and decreased brain pathology 12,14. The cognitive effects of KE have never been tested 
systematically in humans, although there is one case reported in literature in which KE subjectively 
improved cognition in AD 10. Therefore, it is possible that the KE-group in our study might 
experience a transient boost in cognition and mood while on the KE drink.
The positive effects described above were described for the period during which the animals or 
individuals were on the intervention; thus, the potential benefits refer to short-term benefits only. 
No potential long-term benefits are currently known. 
2.3.3
Assessment of Potential Risks and Benefits
Known side effects of consuming KE are common GI tract side effects such as nausea. These have 
been systematically assessed mainly in studies of 1, 4, 5 or 28 days 24,39,40 23,25,26,32,36-38. 
Importantly, it has been shown that when KE is ingested with water, it is well tolerated, especially 
when the dose is ~ 25 g per drink. The most common GI side effect is mild nausea, which is still 
very rare (occurred in 6 out of 2,016 total drinks in one study) 25. In one study authors reported 
frequent GI effects, but these were attributed to large amounts of milk that the supplement was co-
administered with 36. Since the GI side effects may partially be explained by the bitterness of the 
KE drink, we will advise participants to drink some water immediately after the drink to mask the 
bitterness of the supplement.
The terms “ketones”, “ketosis”, “ketogenic” should not be confused with “ketoacidosis”, a 
complication of Diabetes Mellitus during states of a profound imbalance between insulin 
availability and requirements (e.g. during infection, or insulin dose skip). Studies performed in 
healthy individuals have shown that KE supplementation decreases PH, sometimes in the range of 
normal PH and other times at levels of mild acidosis 24,26 . Importantly, in one of those studies, the 
KE supplementation did not have any effect on PH during maximal exercise intensities despite 
decreased PH before exercise 24.  However, supplementation with KE drinks is not comparable to 
Diabetic Ketoacidosis, since KE can increase blood ketones only to a certain degree (which is 
known to be safe) and they drop to baseline within 4 hours 25. Thus, ketosis induced with KE drinks 
is transient and not uncontrolled. On the other hand, insulin deficiency in DKA creates a potentially 
uncontrollable state that will not reverse until insulin requirements are met. Our study population 
will be individuals meeting criteria for MetS which include fasting plasma glucose ≥100 mg/dL or 
drug treatment for high blood glucose 41. However, as an additional layer of safety, we will not 

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include individuals with DM and thus eliminate any possibility of a participant developing 
ketoacidosis due to complete insulin deficit. 
KE and Placebo drinks will be isocaloric. In both groups, individuals will consume 140 x 3 = 420 
calories derived from the supplement, per day. For both groups, we don’t expect any changes in 
systemic metabolic parameters from this amount of calories and that duration of study. Notably, 
KE has been shown to decrease hunger and ghrelin after a single dose 32. However, in a 28-day 
study on the KE drink there was no change in glucose, cholesterol, TGs and body weight 25. The 
placebo will contain dextrose (DXT). Importantly, we will exclude diabetics from the study; 
therefore, any metabolic risk from receiving a supplement containing DXT will be minimal. For a 
typical diet of 2000-2500 calories/day, the 420 calories from either KE or DXT will constitute ~ 
20% of the daily calories. We believe that participants will self-regulate reducing their food intake 
given the intake of these extra calories from the supplement.  
 The value of the information to be gained from this study outweighs the potential risks for the 
following reasons: 
o
The risks are minimal.
o
Individuals may have some transient benefits, such as increased subjective sensation 
of energy, mood and cognitive function.
o
Most importantly, the study will provide proof of concept on whether oral KE 
supplementation can robustly increase brain availability of ketones, in a population 
relevant to AD in terms of brain IR and brain glucose hypometabolism. It will also 
provide evidence for the effects of this intervention on neuronal metabolism and AD-
related cascades (as reflected on EV biomarkers of IR, autophagy, and AD). 
Additionally, the study will provide information on the cognitive effects of KE, 
measured with a sensitive neuropsychological battery.
o
Ultimately, this study could become a “bridge” towards a future clinical study on the 
effects of KE in AD. The importance of this is potentially profound, as there is no 
effective treatment of AD.
3
OBJECTIVES AND ENDPOINTS
OBJECTIVES
ENDPOINTS
JUSTIFICATION FOR 
ENDPOINTS
Primary
To investigate the change in 
brain concentration of BHB, 
using brain MRS, after 28 days 
of supplementation with the KE 
drinks compared to baseline and 
placebo. 
To detect with brain MRS, a 
significant change in the 
concentration of BHB, after 28 days 
of supplementation with KE drinks 
compared to baseline and placebo. 
A previous study on a 
different ketogenic 
intervention (MCTs) given to 
individuals with AD, showed 
that the combined AcAc and 
BHB CMR measured 
globally or at specific 

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OBJECTIVES
ENDPOINTS
JUSTIFICATION FOR 
ENDPOINTS
regions (frontal lobe, parietal 
lobe, temporal lobe, occipital 
lobe, cingulate cortex, 
subcortical regions) with 
PET scan, was significantly 
increased. With this 
ketogenic intervention there 
was also improvement in 
only 3 out of the 23 different 
cognitive tests 6. We expect 
to demonstrate a change in 
brain BHB with KE using 
brain MRS, a technique that 
measures concentration 
rather than CMR.
Secondary
To assess whether genetic 
factors modulate the response to 
the KE supplement.
To assess whether genetic factors 
modulate the response to the KE 
supplement. 
In a previous randomized 
clinical trial of 5:2 
Intermittent Fasting versus 
Healthy Living diet in older 
individuals with insulin 
resistance, we showed that 
the diets had differential 
effects on induction of 
peripheral ketosis based on 
genetic polymorphisms for 
the SLC16A7 and APOE 
genes.35 We expect to find 
similar differential effects on 
induction of peripheral and, 
by extension, brain ketosis in 
response to the KE 
supplement.
Exploratory
i) (a) To investigate the  acute 
effects (single dose) of KE drink 
on brain BHB measured with 
brain MRS, compared to 
i) (a) To detect with brain MRS, a 
significant change in the 
concentration of BHB, acutely after 
single dose of KE drink, compared to 
i) (a) and (b): We have 
shown higher 
Glucose/Creatine and 
Lactate/Creatine 

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OBJECTIVES
ENDPOINTS
JUSTIFICATION FOR 
ENDPOINTS
baseline and placebo;   (b) To 
investigate the  acute (single 
dose) and chronic effects (28 
days on intervention) of other 
brain metabolites measured with 
MRS, compared to baseline and 
placebo); (c) similarly, to 
investigate with MRS, chemical 
changes in the thigh muscle at 
rest, during and after exercise, 
reflecting mitochondrial 
function.
ii) To investigate the acute 
(single dose) and chronic (28 
days) effects of KE drinks 
intake compared to baseline and 
placebo, on blood and urine 
BHB
baseline and placebo. (b) To detect 
with brain MRS, a significant change 
in the concentration of other 
metabolites (glucose, lactate etc), 
acutely after single dose, and 
chronically after 28 days on the KE 
drink compared to baseline and 
placebo. (c) To look with 31P MRS 
(optional) for changes in 
intramuscular pH, ATP and Creatine 
Phosphate in the thigh muscle, 
acutely after a single dose, and 
chronically after 28 days on the KE 
drink compared to baseline and 
placebo. 
ii) Blood will be drawn before and 
after a dose of KE drink or Placebo, 
during Visits 2,3 and 4. Urine will be 
collected during Visits 2 and 4 (over 
~8 hours).
concentration in the 
precuneus of AD patients 
compared to younger and 
older cognitively normal 
participants 2. Decreasing 
these parameters suggests 
improved brain metabolism, 
with less reliance on 
glycolysis, which is linked to 
AD pathogenesis 42. (c) In 
animal models treated with 
ketogenic diets or BHB, 
there is greater energy 
production from ATP 
hydrolysis, improved 
mitochondrial biogenesis, 
metabolomics changes and 
changes in muscle 
morphology 12,43,44. We will 
use 31P MRS to study 
changes in muscle 
bioenergetics with KE 
drinks, which may provide 
additional broad insights on 
the effects of ketones on 
cellular metabolism and 
health.
ii) With the exception of one 
human 28-day study, all 
studies on KE drinks have 
been conducted for a period 
of 5 or fewer days. 
Importantly, the 28-day 
study was designed as single-
arm study without control 
group 33. Thus, the present 
study will confirm the 
efficacy of KE drinks in 
terms of inducing ketosis, by 
measuring BHB in blood and 
urine.

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OBJECTIVES
ENDPOINTS
JUSTIFICATION FOR 
ENDPOINTS
iii) To investigate the chronic 
(28 days on intervention) effects 
of  KE drinks intake on plasma 
EV biomarkers related to insulin 
signaling and resistance and 
autophagy, neuronal and 
astrocytic ketone metabolism, 
AD pathogenic cascades, 
compared to baseline and 
placebo. 
iv) To investigate the acute 
(single dose) and chronic (28 
days) effects of KE drinks 
intake compared to baseline and 
placebo on cognitive 
performance by using scalable 
iii) EV endpoints: 
•
Similar total levels and 
increased levels of activating 
phosphorylations at the level 
of IR, IRS-1, IGF1R, 
canonical cascade (Akt, 
GSK3B, S6K), 
alternative/mitogenic cascade 
(MAPK, JNK, p38), and 
mTOR/p-mTOR 
•
Increased levels of MCT-1 
and 2, and other enzymes 
facilitating ketone 
metabolism 
•
Elevation of NAD+/NADH 
ratio 
•
Aβ40, Aβ42, p181-tau and 
total tau
•
Complement and other 
neuroinflammatory mediators
•
Markers for diverse AD 
pathogenic processes (such as 
lysosomal dysfunction, 
cellular stress responses, and 
other factors that may be 
identified in the future) 
•
RNA species involved in AD 
pathogenesis.
iv) PACC total composite z-score 
and NIH toolbox cognitive 
composite score 
iii) These EV-based 
biomarkers have shown 
significant moderate 
magnitude differences 
between active and placebo 
groups in previous clinical 
trials assessing metabolic 
interventions 45,46. These 
measures are expected to 
change in association with 
change with brain BHB 
measured by MRS, serum 
BHB, and cognitive 
performance.
iv) These composite scores 
assess cognition globally and 
contain measures sensitive to 
detect changes in this 
cognitively normal 
population.

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OBJECTIVES
ENDPOINTS
JUSTIFICATION FOR 
ENDPOINTS
and challenging cognitive 
measures with relevance to AD.
v) To investigate the chronic 
effects of 28 days of KE drinks 
compared to baseline and 
isocaloric placebo supplement 
on gut microbiota and intestinal 
permeability
v) Stool samples will be subjected to 
microbiome analysis by RNA 
sequencing to determine changes in 
bacterial species composition 
between and within groups.
v) Diet has a significant 
impact on gut microbiota.47 
Short-chain fatty acids such 
as butyric acid are the natural 
end-product on non-
digestible carbohydrate 
fermentation by the gut 
microbes and has been 
shown to regulate ketone 
body metabolism during the 
fasted state in mice.48  In 
addition, ketogenic diets 
have been reported to 
produce changes in gut 
microbiota, and differences 
in gut microbiota profile 
after ketogenic diet treatment 
have been reported between 
responder and non-responder 
in epileptic patients.49,50  
Furthermore, age-associated 
changes in gut microbiota 
promote intestinal barrier 
impairment and subsequent 
systemic inflammation and 
metabolic dysfunction in 
mice.51 Therefore, we also 
plan to investigate whether a  
KE drink has effects on gut 
microbiota and intestinal 
permeability.
4
STUDY DESIGN
4.1
OVERALL DESIGN
Study site: The study will take place at a single site and specifically at the NIA Clinical Unit at 
Medstar Harbor Hospital in Baltimore. 
Hypothesis: We hypothesize that acute (single dose) and chronic (28 days) oral administration 
of a KE drink  in cognitively intact middle-age individuals with Metabolic Syndrome (MetS) will 
i) increase peripheral and brain ketone (BHB, AcAc) levels, ii) improve neuronal/astrocytic 

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insulin resistance (IR), induce a switch towards neuronal/astrocytic ketone metabolism, induce 
autophagy and modulate AD pathogenic cascades (as reflected on MRS measures of glycolytic 
metabolism and neuronal/astrocytic EV biomarkers), and iii) improve cognitive performance. 
The changes in MRS and EV biomarkers and cognition will be associated with the level of 
induction in brain ketone levels.   
Study phase: This study is a clinical trial with which we aim to provide proof of concept that 
acute (single dose) and chronic (28 days on intervention) oral KE drinks administration results in 
increased brain’s ketone levels compared to baseline and placebo (containing DXT), which 
correlate with change in MRS and EV biomarkers and cognitive outcomes.
Design: The study will be designed as a double-blinded randomized placebo-controlled trial. 
Study groups and duration of intervention: The study will involve two groups: KE drink vs 
placebo (DXT). The duration of the intervention will be 28 ± 3 days. 
Name of study’s intervention: (R)-3-hydroxybutyl (R)-3-hydroxybutyrate, which is formulated 
as a drink, the KE drink
Dose of intervention: 25 g of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate contained into a water-
based drink x 3 times per day x 28 days. 
Dose escalation: Not planned
Interim analysis: Not planned
Stratification:  Randomization will be stratified by age. There will be 2 groups defined as 
participants age 55-64 and participants ≥ 65 years old).
Sub-studies: Not planned
4.2
SCIENTIFIC RATIONALE FOR STUDY DESIGN
Our intervention aims to increase the energy supply to the brain by providing exogenous ketones 
in the form of an ester that is contained in a drink and thus improve its function. To assess if the 
brain effects of a KE drink are due to specific mechanisms of actions of ketones and not just due 
to increased caloric supply, we chose to use an isocaloric placebo (DXT). It is common practice 
to use an isocaloric drink for placebo in clinical studies in which a ketogenic compound is tested 
7,21,22,32,37. Several previous human studies on a KE drink implemented an isocaloric placebo based 
on solution of dextrose (DXT) 32,37. 
The placebo group will meet the same eligibility criteria as the intervention group. Our study will 
be designed as double-blinded randomized placebo-controlled clinical trial. The consent form 
refers to both drinks as “supplements” and informs them of the number of calories they contain 
and the fact that one of them contains ketones and the other does not, but not of their names. See 
6.1.1 for description of placebo ingredients.
4.3
JUSTIFICATION FOR DOSE
We will administer the KE drink compound orally since this is the only known route which has 
been tested for this supplement. The chosen dose (25 g of (R)-3-hydroxybutyl (R)-3-

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hydroxybutyrate contained into a water-based drink x 3 times per day) has been decided based on 
its safety, tolerability and efficacy in producing robust peripheral ketosis in previous human studies 
10,25,26,36,38. The duration of intervention (28 days) was chosen such that we can assess the acute 
and chronic effects of KE on brain BHB levels, but also its chronic effects on MRS metabolites 
related to glycolytic metabolism, neuronal/astrocytic insulin resistance (IR), neuronal/astrocytic 
ketone metabolism, autophagy and AD pathogenic cascades (as reflected on neuronal/astrocytic 
EV biomarkers), and on cognitive performance. Furthermore, the 28-day duration of KE drinks 
administration has already been tested in humans in a study during which KE was shown to be 
both tolerable and safe 25.
We predict that brain ketone body concentration (especially BHB) will increase acutely after a 
single dose and within the range of detection for MRS, because brain uptake is proportional to 
blood levels which have been shown to increase acutely after a single dose of KE 23,24,32,37,40,52. 
However, it is likely that the increase in brain BHB after a dose of KE will be more robust after 
having received the supplement for 28 days. Moreover, having received the supplement for 28 
days may be needed to demonstrate a (i) shift to the favorable direction of EV biomarkers reflecting 
neuronal and astrocytic metabolism and function, (ii) improvement in cognitive performance, (iii) 
improvement in mitochondrial function in muscle, and (iv) change in gut microbiome.
We conclude that a 28-day period on the proposed dose would be both safe and tolerable and 
would also be a meaningful length of time for assessing change in biomarkers and cognitive 
performance.
5
STUDY POPULATION
5.1
INCLUSION CRITERIA
In order to be eligible to participate in this study, an individual must meet all of the following 
criteria:
1. Ability to provide informed consent and willingness to sign a written informed consent 
document
2. Male or female, age ≥ 55 years old 
3. Cognitively intact status ascertained during screening (defined as absence of significant 
memory or cognitive changes in the last 2 years by subjective report, Clinical Dementia 
Rating (CDR) of 0, and Montreal Cognitive Assessment (MoCA) ≥  26) 
4. Ability to take oral medications 
5. Willingness to adhere to all study procedures including having MRI/MRS.
6. Presence of Metabolic Syndrome (MetS). Specifically, they should meet three of the five 
following MetS diagnostic criteria to be eligible 53:
•
Receive drug treatment for elevated triglycerides (TGs) or have serum TGs ≥ 150 
mg/dL (1.7 mmol/L)
•
Receive drug treatment for low HDL-cholesterol or have serum HDL-cholesterol 
<40 mg/dL (1.0 mmol/L) in males; <50 mg/dL (1.3 mmol/L) in females
•
Receive drug treatment for high Blood Pressure (BP) or have BP ≥130/85 mmHg 
•
Receive drug treatment for high blood glucose or have fasting plasma glucose 
≥100 mg/dL

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•
Central obesity, defined as a waist circumference ≥102 cm (40 in) in men and ≥88 
cm (35 in) in women 41.
5.2
EXCLUSION CRITERIA
An individual who meets any of the following criteria will be excluded from participation in this 
study:
1. Previously diagnosed with a condition causing clinically significant cognitive impairment, 
such as MCI, AD or other type of dementia (such as vascular dementia, Lewy body 
dementia and frontotemporal dementia).
2. History of clinically significant brain disorders, such as stroke, multiple sclerosis, 
Parkinson’s disease or other movement disorders, brain tumors, history of meningitis or 
encephalitis, history of moderate or severe traumatic brain injury (defined as Glasgow 
Coma Scale of 12 or less), epilepsy. Certain common neurological disorders not considered 
relevant (e.g. migraine, essential tremor) or incidental neuroimaging findings that are 
common and of uncertain clinical significance (e.g. mild-moderate microvascular changes 
on MRI) may be allowed.
3. Chronic and significant psychiatric conditions (e.g. history of bipolar disorder, 
schizophrenia, PTSD, moderate to severe depression or treatment-resistant depression. 
Unipolar depression or anxiety disorder may be allowed if mild or if successfully treated 
with single anti-depressant or anti-anxiety agents. 
4. Positive urine drug screen (and no prescription medication accounting for the positive test). 
5. Positive HIV, HBV or HCV status during screening.
6. Contraindications for MRI (pregnancy, pacemakers or other implanted devices, ferrous 
metal implants or shrapnel in or around the head etc.).
7. Anemia (defined as HGB < 12 for men or < 11 g/dl for women)
8. Poor venous access.
9. Lactation or Pregnancy (positive urine pregnancy test. Pregnancy tests will not be done on 
post-menopausal women defined as one of the below criteria:
a.
prior bilateral oophorectomy 
b. Amenorrhea for 12 months or more
10. Known severe allergic reactions to the KE drinks or other ketogenic supplement or stevia 
products.
11. Following high fat/low carb diet (ketogenic) diet or very low calorie (<500 calories) diet 
or taking other ketogenic supplements (such as Medium Chain Triglycerides (MCTs), 
Ketone Salts) or fasting intermittently and unwilling to stop it while on the KE 
drink/Placebo.
12. Very high or severe hypertriglyceridemia (≥886 mg/dL or 10.0 mmol/L)
13. Severe Hypertension (systolic blood pressure ≥180 mmHg and/or diastolic blood pressure 
≥120 mmHg)
14. Weight ≥ 300 lbs (MRI scanner weight limit)
15. Diabetes Mellitus (type 1 or 2)
16. Taking the drug metformin.
17. Non-English speakers (given staffing constraints for cognitive testing administration and 
need for decreased variability in testing procedures for a small N study).

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18. Participant has any concurrent medical condition, so that participation in the clinical study 
would not be in her/his best interest, in the PI’s judgement. 
19. To be eligible to consent for optional thigh MRI: Individuals with joint replacements that 
may be affected by the defined exercise protocol or which may prevent MRI analysis or 
any condition, in the opinion of the investigator, that would prevent successful completion 
of the exercise protocol such as, but not limited to reported osteoarthritis, rheumatoid 
arthritis and/or fibromyalgia. 
5.3
INCLUSION OF VULNERABLE PARTICIPANTS
•
Children: No inclusion is planned. This study aims to investigate the effects of aging (≥ 
55 years old) on brain metabolism, therefore inclusion of children is inappropriate with 
respect to the purpose of the research.
•
Pregnant Women, Fetuses, or Neonates: No inclusion is planned. We will exclude 
women, fetuses or neonates with respect to the health of participants, specifically, due to 
the risk associated with the MRI procedure and the unknown effects of the ketone ester 
supplement. 
•
Prisoners: No inclusion is planned. This study involves several visits to the NIA Clinical 
Unit, which prisoners are unable to perform. Therefore, inclusion of prisoners is 
inappropriate with respect to the purpose of the research.
•
Decisionally Impaired Adults: No inclusion is planned. Cognitive intact status will be 
ascertained at baseline based on Clinical Dementia Rating (CDR) of 0, and Montreal 
Cognitive Assessment (MoCA) ≥  26, since the main objective of the study is to study a 
population of cognitively intact individuals. Therefore, inclusion of decisionally impaired 
adults is inappropriate with respect to the purpose of the research. Moreover, participants 
are not expected to become decisionally impaired over the course of the study. 
•
NIH Employees: We intend to include NIH staff (NIH contractors and special 
volunteers, guest researchers and trainees) and family members of study team members 
in this study. The staff and family members will be invited to participate through standard 
recruitment efforts. Solicitation of subordinates will not occur by direct contact. 
Recruitment will occur as per our recruitment plan in section 4.2.  Participation will be 
voluntary and neither participation nor refusal to participate will have an effect, either 
beneficial or adverse, on the participant’s employment or position at NIH. Procedures 
will be followed in accordance with NIH HRPP Policy 404 Research Involving NIH Staff 
as Subjects.
For NIH employees:
o
Neither participation nor refusal to participate will have an effect, either beneficial 
or adverse, on the participant’s employment or work situation.
o
We will ensure all employees review the “NIH Frequently Asked Questions 
(FAQs) for Staff Who are Considering Participation in NIH Research”.
o
NIH Staff will also be asked to review the Leave policy for NIH Employees 
Participating in NIH Medical Research Studies (NIH Policy Manual 2300-630-3).

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o
The employee subject’s privacy and confidentiality will be preserved in 
accordance with NIH Intramural policies, which define the scope and limitations 
of the protections.
o
Subjects that are employees/staff will be consented in the usual manner. The 
inclusion of employees or staff is not anticipated to affect the research outcome; 
therefore, if the subject is a co-worker (including a supervisor, subordinate or 
coworker) the subject may be consented by another co-worker and/or subordinate. 
The study PI will not be involved in the consenting of employees or staff.
•
Other vulnerable populations: No inclusion is planned. 
5.4
LIFESTYLE CONSIDERATIONS
During this study, participants will be asked to refrain from: high fat/low carb diet (<50 g carbs/d) 
diet or very low calorie (<500 calories) diet or other ketogenic supplements/foods (such as Medium 
Chain Triglycerides (MCTs), Coconut oil, Ketone Salts), or intermittent fasting.
5.5
SCREEN FAILURES
Screen failures are defined as participants who consent to participate in the clinical trial but are 
not subsequently assigned to the study intervention or entered in the study. A minimal set of screen 
failure information is required to ensure transparent reporting of screen failure participants, to meet 
the Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to 
respond to queries from regulatory authorities. Minimal information includes demography, screen 
failure details, eligibility criteria, and any serious adverse event (SAE).
Individuals who do not meet the criteria for participation in this trial (screen failure) because of a 
“positive urine drug screen” may be rescreened in the future. Rescreened participants should be 
assigned the same participant number as for the initial screening. 
5.6
STRATEGIES FOR RECRUITMENT AND RETENTION
We aim to recruit and screen up to 150 participants to ensure that 50 participants will meet 
eligibility criteria and complete all study procedures. We expect 5-10 participants to be 
successfully recruited per month and 2-4 of them to qualify and start on a supplement. All 
participants will be recruited in the US. The source of participants will be the general public. 
Recruitment will be done primarily through advertisement in newspapers, hardcopy and electronic 
media, distribution and posting of flyers and through NIH-approved website postings. 
Procedures that will enhance adherence to the intervention and participant retention are: 
o
Phone calls - once per week during non-visit weeks. During the calls, we will discuss 
compliance and potential concerns or questions that participants may have; we will also 
elicit symptoms and potential AEs. 
o
The study will involve a visit (Visit 3) on the Day 14 ± 3 of the intervention, to assess 
compliance and reinforce motivation. Peripheral blood ketone levels will be measured. 

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o
Participants will be advised to keep a log on a daily basis in which they will report the 
timing of the KE self-administration. They will be advised to bring that log on the 
compliance and final Visits (Visits 3 and 4). 
5.6.1
Costs
None expected.
5.6.2
Compensation
All participants will be compensated for their time and research-related inconveniences via NIA 
debit cards. For participants that are NIH employees, we will follow NIH HRPP Policy 404. 
Payments will be given as stated below. Compensation will typically be prorated for parts 
completed if the participants do not complete the study. Participants will be compensated for each 
visit as follows:
Visit 1 (Screening)
$50
Visit 2 (Baseline and Acute Effects)
$300
Visit 3 (Compliance Assessment)
$200 
Visit 4 (Chronic Effects)
$500
Visit 5 (blood draw for DNA collection)
$50
Compensation will be $1100 for participants that complete the study. Participants will receive 
additional $20 for each optional thigh muscle MRS completed (up to four thigh muscle MRS for 
the duration of the study or additional $100). If they do not complete the study, they will be paid 
for those visits or parts completed:
Compensation per visit: as above
Compensation per part completed:
•
blood draws (per session) $25; if two sessions are completed per visit, $50 
•
blood draws (for DNA) $50
•
brain MRI/MRS (per session) $50; if two sessions are completed per visit, $100
•
optional Thigh MRI (per session) $20; if two sessions are completed per visit, $40
•
cognitive testing $50, 
•
Stool sample collection $ 10
•
Urine sample collection $ 10
•
Participants will be offered snacks 
•
KE/Placebo for one week $50
and a meal during Visits 1, 2, 3, 4 and 5.
No travel or lodging compensation will be provided, because all subjects will be recruited from 
the greater Baltimore area.  

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6
STUDY INTERVENTION
6.1
STUDY INTERVENTIONS(S) ADMINISTRATION
6.1.1
Study Intervention Description
The KE substance is regulated under Generally Recognized as Safe (GRAS) by FDA 
(https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=515) and is being 
advertised and sold as an oral supplement . The investigation is not intended to support any change 
in advertising of the supplement or to support an indication for use as treatment for any disease. 
Also, the investigation does not involve any different than the original route of administration of 
KE (oral). The amount of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KE) per dose will be 25 g. 
Also, the daily dose and duration of the intervention that will be used, have already been tested in 
previous clinical trials 10,25,26,36,38..  The KE drinks will be repackaged by the NIA Pharmacist into 
new bottles identical to the ones that will be used for the placebo to ensure the blinding of 
participants and researchers to the drink.
The main content of the Placebo will be an aqueous solution containing ~ 35 g of DXT. To better 
match the taste of the placebo to that of the KE drink, we will add a fruity flavor powder and stevia. 
We will also add Denatonium Benzoate (Bittrex) to match the bitterness of KE drink. The placebo 
will be prepared and dispensed by the NIA Pharmacist.
6.1.2
Dosing and Administration
The dose and formulation (25 g of KE - (R)-3-hydroxybutyl (R)-3-hydroxybutyrate contained in 
the KE drink), daily scheme (3 times daily) and total duration (28 days) are based on previous 
human studies on this dose and will be identical for all participants in the KE group throughout 
the study without dose changes 25. The placebo (the main ingredient of which will be (~ 35 g of 
DXT) will also be given at an identical dose for every participant of the Placebo group throughout 
the study, providing same calories as the KE drink (140 calories per dose).  
We don’t expect any abnormal laboratory values as a result of the studied intervention that could 
mandate a change in dosing. 
Drug Administration
Ketone Ester drink or isocaloric Placebo will be taken 3 times per day every 6-8 hours; suggested 
times are 8-10 am, 2-4 pm, 8-10 pm, but these may be modified by individual participants at home 
to fit their schedule. Participants will be advised not to take any dose in less than 4 hours from a 
previous dose. Participants will be advised to take the supplement as is and not mix it with any 
other beverage, especially with large amounts of milk. On the other hand, they will be advised to 
consume water after or during the supplement intake to decrease the taste of bitterness (both 
interventions will be bitter, and bitterness could potentially increase the risk of GI side effects). 
If one or more doses are skipped, individuals will be advised to continue with the next dose(s), as 
originally scheduled, and not add “make up” doses  

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6.2
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
6.2.1
Acquisition and Accountability
The KE formulation will be acquired by a Ketone Ester drinks supplier who can formulate drinks 
containing 25 g of the GRAS substance (R)-3-hydroxybutyl (R)-3-hydroxybutyrate. The main 
ingredient of the Placebo (~ 35 g of DXT) is widely available and will be bought from an approved 
NIH supplier. Both products will be received by the NIA Pharmacy and will be stored and 
repackaged in identical new bottles by the NIA Pharmacist. In addition, the Pharmacist will add 
Denatonium Benzoate (Bittrex) and stevia (both bought from an approved NIH supplier), and 
fruity flavor to match the taste of KE drinks.
6.2.2
Formulation, Appearance, Packaging, and Labeling
Ketone Ester (KE) drinks:
Ketone Ester drink is a commercially available beverage, sold as a “sports drink” in the USA since 
November 2017. One serving of the KE drinks  contains 25g of ketone ester (KE- (R)-3-
hydroxybutyl (R)-3-hydroxybutyrate), with the remaining volume being water, flavors and 
preservatives. 
The KE was previously determined to be Generally Recognized as Safe (GRAS) by the US FDA 
(GRN No. 515) allowing it to be marketed as a food ingredient. It is produced via an enzyme- 
catalyzed transesterification reaction of ethyl-D-hydroxybutyrate and (R)-1,3-butanediol. 
Placebo: 
The Placebo will consist of ~ 35 g of DXT, bitter taste (Bittrex)and additional flavors. Isocaloric 
placebos are commonly used in ketogenic interventions with cognitive and other functional 
outcomes 7,21,22,32,37. Moreover, whenever the HVMN KE supplement has been compared to an 
isocaloric placebo, the latter has been DXT 32,37.
6.2.3
Product Storage and Stability
According to communication with a KE drink supplier, the KE drinks can be stored in room 
temperature. The stability of the KE mixed with water (1:1) at different temperatures and different 
pH levels has been assessed over a 330-day period. KE generally remained stable when stored at 
temperatures ranging from 4 to 37°C and pH levels ranging from 3 to 10 throughout the storage 
period. However, because of the repackaging of the KE drinks into new bottles by the NIA 
Pharmacist, we will advise the participants to keep the drink refrigerated, before they consume it. 
Similarly, we will advise the participants to keep the placebo drink (see 6.2.4 details on its 
preparation by the NIA Pharmacist) refrigerated before consumption.
Both KE drinks and Placebo formulations that participants will take, will be prepared and 
dispensed by the NIA Pharmacist. Both the repackaged KE drinks and Placebo will be stored 
refrigerated. Sufficient supplies for KE drink or placebo drink for 14 +3 days (51 doses) will be 
given during Visit 2 and additional supplies for 14+3 days (51 doses) will be given during Visit 3. 
Participants will be asked to return any unused doses. 

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KE preparation: the KE product will be repackaged into new bottles to ensure a double-blind 
design. 
Placebo preparation: for placebo, the Pharmacist will prepare an aqueous solution of DXT, fruity 
flavor concentrate, stevia and bitterness (Bittrex)to match the bitter flavor of KE drinks and be as 
palatable as possible. The solution will be packaged into identical bottles to those used for the KE 
drinks.
6.3
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING
This study will be a double-blinded randomized controlled placebo trial. 
To avoid selection bias, eligible participants will be randomly allocated in 1:1 ratio, to either oral 
intake of  KE drink or Placebo, using a computer based random sequence generation method which 
will be concealed. Randomization will be stratified by age. There will be 2 groups defined as 
participants age 55-64 and participants ≥ 65 years old. The formulation of KE drinks and Placebo 
will be done in a way that does not allow participants to find out in which group they are assigned 
to. Efforts to match the Placebo with KE drinks are described in 6.2.3.
Unintentional break of the blinding of participants will be reported to the IRB as a study deviation.
Credibility expectancy questionnaire (CEQ): 
To assess subjects expectations and confirm their blind status, we modified the CEQ, a tool 
commonly used in studies of chronic pain and anxiety syndromes 54-56. The CEQ will be 
administered on Visits 3 and 4 (i.e., while participants are receiving the study supplement). It has 
been shown that expectations can alter the outcome of a therapy in diverse populations and 
conditions, such as Vietnam veterans, social phobia and generalized anxiety disorder; thus, the 
CEQ is a valuable questionnaire to exclude that any therapy outcomes are explained by 
expectations. In our study, participants do not have a disease or cognitive impairment. 
Nevertheless, cognitive performance, an important exploratory outcome, will be assessed and it is 
conceivable that it may be affected by expectations. Therefore, responses will be compared 
between groups and may be used as covariate in sensitivity analyses for cognitive outcomes.
The questions that will be asked are: 
•
“At this point, how effective do you think the supplement you are receiving will be 
in changing your brain chemistry and improving your cognitive function”
•
“How confident would you be in recommending the supplement to a friend who 
wishes to change her/his brain chemistry and improve her/his cognitive function”
•
“By the end of the 28-day period, how much change in your brain chemistry and 
improvement in your cognitive function do you think will occur”?
•
“At this point, how much do you really feel that the supplement you are receiving 
will help you to change your brain chemistry and improve your cognitive function”
•
“By the end of the therapy period, how much change in your brain chemistry and 
improvement in your cognitive function do you really feel will occur”

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Participants will record their responses on a 9-point Likert scale from 0-3 (“Not at all”) to 
4-6 (to some extent) to 7-9 (“Very much”) in some questions, and as a percentage (0-100 %) in 
other question.  
6.4
STUDY INTERVENTION COMPLIANCE
For the assessment of compliance, we will take the following measures:
•
Visit to assess compliance (Visit 3): We will assess compliance during Visit 3, which will 
take place in between Visits 2 (baseline and assessment of acute effects) and 4 (assessment 
of chronic effects) of the study. During this Visit, blood will be drawn for the assessment 
of blood BHB levels. In addition, we will assess their compliance logs (see below).
•
Phone calls: We will place phone calls once between Visits 2, 3 and once between Visits 
3, 4. Potential concerns and/or questions will be discussed in the calls and symptoms/AEs 
will be elicited.
•
Logs of supplement intake: Participants will be asked to keep a log on which they will 
report the timing of each dose of the drink they take. Keeping a log will reinforce their 
commitment to take the supplement as instructed properly. They will bring in these logs 
during Visits 3 and 4.
6.5
CONCOMITANT THERAPY
For this protocol, both prescription medications (defined as a medication that can be prescribed 
only by a properly authorized/licensed clinician) and over-the-counter medications and 
supplements are potentially important modifiers of the effect of the interventions. Therefore, 
concomitant prescription medications, over-the-counter medications and supplements will be 
recorded during all four planned Visits. 
Excluded concomitant compounds will be ketogenic supplements such Medium Chain 
Triglycerides, Coconut oil, Ketone Salts or any other supplement advertised as “ketogenic”.
We do not expect any interactions of any of the allowed concomitant medications with the study 
intervention. Also, allowed concomitant interventions are not expected to affect any of the study 
outcomes.  
7
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
7.1
DISCONTINUATION OF STUDY INTERVENTION
Discontinuation from study’s intervention means discontinuation from the study. If a clinically 
significant finding is identified (including, but not limited to changes from baseline) after 
enrollment, the PI will determine if any change in participant management is needed. Any new 
clinically relevant finding will be reported as an adverse event (AE).
Participants are free to discontinue the study intervention at any time upon request.

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7.2
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY
Participants are free to withdraw from participation in the study at any time upon request. 
The PI may discontinue a participant from the study for the following reasons:
•
Screen failure.
•
The participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation
•
The participant has an adverse event (AE), including clinical signs and/or symptoms, 
laboratory abnormality, or other medical condition or situation, such that continued 
participation in the study would not be in the best interest of the participant, at the 
judgment of the PI.
•
The participant manifests significant study non-compliance (e.g.  the participant was 
deemed eligible but didn’t show for any Visit. Also, if the participant’s log of supplement 
intake during Visit 3 shows multiple skipped doses or skipped days of intervention).
•
The participant has any condition, which in the opinion of the PI, could interfere with the 
study procedures.
•
MRI will be stopped at the request of the participant as a result of claustrophobia or 
problems associated with lying in the scanner for any length of time. If MRS measures for 
BHB (primary outcome) cannot be obtained, this will constitute discontinuation of the 
participation.
•
Death.
Participants that are discontinued/withdrawn from the study will be replaced by new participants. 
The reason for participant discontinuation or withdrawal from the study will be recorded on the 
medical record. 
7.3
LOST TO FOLLOW-UP
A participant will be considered lost to follow-up if they do not present in one or more Visits. The 
following actions will be taken if a participant fails to return to the clinic for a required study Visit:
•
The site will attempt to contact the participant and reschedule the missed Visit. 
Specifically, for Visit 4, which is the final Visit, we will attempt to reschedule it within 3 
days from the original date. For Visit 1 (baseline Visit) and Visit 2 (initiation Visit), the 
participants can reschedule according to their availability. For Visit 3 (compliance Visit), 
they should reschedule within the 3 next days.
•
Before a participant is deemed lost to follow-up, the investigator or designee will make 
every effort to regain contact with the participant (where possible, place 3 telephone calls 
and, if necessary, a certified letter to the participant’s last known mailing address). These 
contact attempts should be documented in the participant’s medical record. 
•
Should the participant continue to be unreachable, she/he will be considered to have 
withdrawn from the study with a primary reason of “lost to follow-up”.

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8
STUDY ASSESSMENTS AND PROCEDURES
8.1
SCREENING PROCEDURES
The screening procedures will involve two phases: first we will contact individuals who are 
interested in participating in the study via phone and pre-screen them according to their self-report 
of meeting inclusion/exclusion criteria. Next, if not excluded via phone pre-screening, we will 
screen them during Visit 1 to ascertain inclusion/exclusion criteria. No screening procedures will 
take place until the subject has provided informed consent during Visit 1. During the informed 
consent, participants will be given the option to participate in the additional thigh muscle MRS 
study. If they opt to not participate in it, they will still be eligible to complete the rest of the 
procedures. The determination of eligibility will involve the next procedures:
•
Nursing Assessment
•
Assessment of vital signs and anthropometric measures (waist circumference, etc.),
•
Blood drawing for screening clinical labs: complete blood count (CBC) with automatic 
differential, lipid panel (including HDL and TGs, which are part of the definition of MetS), 
complete metabolic panel (including glucose, which is part of the definition of MetS), 
HgbA1c, and fasting insulin, HIV, HBV, HCV.
•
History and Physical
•
Urine test for drugs
•
Urine pregnancy. Pregnancy tests will not be done on post-menopausal women defined as 
one of the below criteria:
a.
prior bilateral oophorectomy 
b. Amenorrhea for 12 months or more
•
Cognitive assessment for eligibility (CDR, MOCA)
8.2
EFFICACY ASSESSMENTS
8.2.1
Clinical Evaluations
No clinical evaluations are meant to assess efficacy.
8.2.2
Neuropsychological Assessment
Cognitive testing will be administered by a study investigator during the screening, baseline and 
final visits (Visits 2 and 4). Screening cognitive testing will consist of screening tests that are 
commonly used to ascertain normal cognitive status: participant-informed Clinical Dementia 
Rating (CDR) scale (global score = 0) and Montreal Cognitive Assessment (MoCA) (≥  26). For 
deriving efficacy measures, we will use a battery modeled after the preclinical Alzheimer cognitive 
composite (PACC 57), which includes the Free and Cued Selective Reminding Task (FSCRT) for 
list learning, the Logical Memory task for episodic memory, the Digit-Symbol task for timed 
executive function, the MoCA as a global cognitive measure, and an overall Z-transformed 
composite score. In addition, attention, episodic memory, working memory, language/fluency, and 
executive function, and their composite score will be assessed via the NIH Toolbox 
(www.nihtoolbox.org 
58); a comprehensive, psychometrically sound, and well-validated 
computerized testing battery that reliably and uniformly assesses cognitive function. Cognitive 

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testing will take place before the supplement intake at Visit 2 (baseline) and ~3 hours after 
supplement intake at Visit 4 to assess acute on chronic effects.
8.2.3
Magnetic Resonance Spectroscopy (MRS)/Magnetic Resonance Imaging (MRI): 
The 3T Philips MRI system used in the NIA Clinical research Unit is an FDA-approved clinical 
device which we use within its marketed purposed for our research. Some of the MRI sequences 
used in this study require that the 3T Philips scanner be run in “Research Mode” using research 
pulse sequences, all of which operate within FDA guidelines, under the International 
Electrotechnical Commission (IEC) 60601-2-33, First Level-Controlled Operating Mode. The 
IEC/FDA define operating mode limits on MRI studies for RF heating (Specific Absorption Rate) 
and Time-varying magnetic field gradients (dB/dt). The research sequences can be classified as 
NSR devices because normal, healthy volunteers are used in this study and these pulse sequences 
adhere to the FDA/IDE safety standards. Therefore, there is no significant risk to the subject from 
these research pulse sequences. Furthermore, research software is used to process and analyze the 
data.  Since the processing does not add any increased risk to the healthy volunteers in this study, 
it is also classified as NSR.  The research pulse sequences and research software will not be used 
for diagnosis or clinical decision making.
This investigational device, as utilized in this study, is considered a Non-Significant Risk (NSR) 
device per 21 CFR 812.2(b)(1).  The NSR classification was established as the device does not 
meet the following criteria for a Significant Risk Device (21 CFR 812(m)), i.e., a device that:
1. Is intended as an implant and presents a potential for serious risk to the health, safety, or welfare 
of a subject;
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;
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; or
4. Otherwise presents a potential for serious risk to the health, safety or welfare of a subject
A standard MRI safety screen is administered prior to participation in any studies involving MRI 
or MRS to verify that participants can safely enter the magnetic area.  
a) Brain: 
Structural brain MRI (3D T1 sequence MPRAGE for voxel placement and partial volume 
correction, T2, DWI, and FLAIR to provide clinical read) and resting state fMRI to associate with 
MRS metabolites as previously done 59.
Single voxel 1H-MRS data will be acquired using a Philips 3T whole-body MR scanner equipped 
with an 8-channel SENSE head coil. A volume of interest of 25 × 18 × 20 mm3 will be placed at 
midline over the bilateral posteromedial cortex (PMC). To accurately measure brain Glucose 
concentration, we will acquire a two-dimensional j-resolved spectrum using a J-PRESS sequence 
with maximum-echo sampling 60. This method acquires a dynamic series of PRESS spectra 
incrementing the echo time to encode J-modulations in the second (indirect) dimension; this 

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additional dimension permits the discrimination and quantification of resonances that overlap in 
the directly detected chemical shift direction but have different J couplings. The echo times in this 
study are to be incremented from 31 ms to 229 ms using 100 echo steps with a step size of 2 ms. 
JPRESS acquisition parameters consist of a repetition time = 1600 ms, eight averages per echo 
time, bandwidth in the direct dimension = 2 kHz, 1024 sample points, for a total scan duration of 
21 min and 20 s. Line widths for the water resonance are monitored for intra-subject scan reliability 
and have been previously observed at a stable (mean ± SD) 7.3 ± 1.7 Hz.
A PRESS sequence with non-suppressed water signal reference in the same region will be acquired 
to obtain measurements of the ketone bodies β-hydroxybutyrate, aceto-acetate, and acetone 34,61. 
A reliable modified basis set including these ketone metabolites was acquired via personal 
communication from Dr. Stephen Provencher in July of 2019. PRESS parameters are: TE = 35 ms, 
TR = 2000 ms, 256 averages, direct dimension bandwidth = 2 kHz, and 2048 sample points. Data 
will be processed with LCModel 62. 
Total scanning time will be ~45 minutes for each brain-scanning session. In both Visits 2 and 4, 
two brain-scanning sessions will take place, first, during fasting, then ~ 90 minutes after 
supplement intake, to assess both baseline and changes after the administration of the KE drink. 
b) Optional thigh Muscle 31P MRS: 
We will implement a moderate leg exercise protocol combined with 31P MRS to assess muscle 
bioenergetic capacity. The exercise protocol consists of knee extension performed while the 
subject is lying supine in the MRI scanner.  The average duration of exercise will be 30 seconds, 
based on two criteria as determined by real-time evaluation of 31P spectra: (i) reduction in 
phosphocreatine (PCr) peak height by 30% to 70% compared with initial values, and (ii) avoidance 
of significant intracellular muscle acidification, defined as pH lower than 6.8.  
All spectra will be obtained using a pulse-acquire sequence, with a 6-second duration for each 
acquisition. Acquisition will be initiated 60 seconds before exercise and continued throughout the 
exercise period, and then for 360 seconds during the post-exercise recovery period. Thus, a total 
of 75 spectra will be obtained for each participant's exercise protocol. The information content of 
these 31P spectra includes intramuscular pH, and PCr and ATP levels relative to their resting level. 
pH is expected to decline during exercise and recover post-exercise, ATP is expected to remain 
constant for this moderate exercise protocol, and PCr is expected to decline during exercise and 
recover post-exercise. The time constant of this post-exercise PCr recovery will be obtained 
through use of a mono-exponential fit of the recovery data and will be expressed as τPCr, in units 
of seconds. This time constant is inversely proportional to maximum muscle oxidative capacity, 
with a slower recovery, indicated by a larger τPCr, reflecting lower oxidative capacity.
Total scanning time (including set up) will be ~ 45 min for each muscle 31P MRS session. In Visit 
2 and 4, two muscle-scanning sessions will take place, first, during fasting, then ~ 1.5 hours after 
the administration of the KE drink.
8.2.4
Compliance Assessments
In addition to including a dedicated compliance visit (Visit 3), we will communicate with 
participants via phone call, once between Visits 2, 3 and once between Visits 3, 4. Participants 
will also be advised to keep a log of the times of taking each dose. They will be asked to bring 
the Log in for review on Visits 3 and 4.

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8.2.5
Biospecimen Evaluations
Biological specimen collection
Clinical labs will be sent to CLIA Medstar Harbor Hospital and results will be acquired in real 
time. All other labs will be done for research purposes and sample processing (e.g. to derive EVs) 
and measurements (e.g. serum BHB, plasma EV biomarkers) will be conducted in batches by 
research Lab personnel at the NIA Biomedical Research Center. HgbA1c will be performed by the 
NIA Harbor Lab. 
Venous blood draws and urine collection during Visit 1 (Screening Visit): 
Up to 40 ml of blood will be drawn for the assessment of screening clinical labs including CBC 
with differential, Comprehensive Metabolic Panel including fasting glucose, Lipids panel (TGs, 
total cholesterol, LDL, HDL), HgbA1c, and fasting insulin, HBV, HCV and HIV. All screening 
blood draws will be performed after fasting for 12 hours. All of these tests will be conducted by 
the CLIA Medstar Harbor Hospital lab except for the HgbA1c which will be performed by the 
NIA Harbor Lab. 
Urine will also be collected to test for pregnancy in women with child-bearing potential. See 
section 8.1.
Venous blood draws and stool/urine samples during Visit 2 (Baseline Visit): 
Up to 40 ml of blood will be collected in (i) serum separator tubes for eventual measurement of 
β-hydroxybutyrate (BHB) by BRC research personnel; and (ii) EDTA tubes for eventual EV 
isolation and biomarkers by BRC research personnel. Up to 35 ml will be collected before the 
first supplement dose and ~5 ml after ~ 60-75 minutes from it. Urine will also be collected to test 
for pregnancy in women with child-bearing potential. See section 8.1. Stool samples (brought 
from home) will also be collected for assessment of gut microbiome before the first dose. The 
stool samples should be collected at home, no more than 24 hours before this Visit. The 
participants will be provided supplies and instructions to obtain the stool sample. Urine will be 
collected throughout the Visit (over ~ 8 hours), for measurement of urine BHB. Urine pregnancy 
test will be also performed in women with child-bearing potential. See section 8.1.
Venous blood draws during Visit 3 (Compliance Visit): 
Up to 40 ml of blood will be collected in (i) serum separator tubes for eventual measurement of β-
hydroxybutyrate (BHB) by BRC research personnel; and (ii) EDTA tubes for eventual EV 
isolation and biomarkers by BRC research personnel. Up to 35 ml will be collected before the 
supplement dose and ~5 ml after ~ 60-75 minutes from it. Urine will also be collected to test for 
pregnancy in women with child-bearing potential. See section 8.1. Urine pregnancy test will be 
also performed in women with child-bearing potential. See section 8.1.
Venous blood draws and stool/urine sample during Visit 4 (Outcome Visit): 
Up to 55 ml of blood will be collected for (i) clinical labs, including CBC with differential, 
Comprehensive Metabolic Panel including fasting glucose, Lipids panel (TGs, total cholesterol, 
LDL, HDL), HgbA1c (will be performed by the NIA Harbor Lab), and fasting insulin (will be 
conducted by the Medstar Harbor Hospital lab); and (ii) serum separator tubes for eventual 
measurement of β-hydroxybutyrate (BHB) by BRC research personnel; (iii) EDTA tubes will be 
drawn for eventual EV isolation and biomarkers by BRC research personnel. Up to 50 ml will be 

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collected before the supplement dose and ~5 ml after ~ 60-75 minutes from it. Urine will also be 
collected to test for pregnancy in women with child-bearing potential. See section 8.1. 
Stool samples (brought from home) will also be collected for assessment of gut microbiome after 
the supplement intake. The stool samples will be collected at home, no more than 24 hours before 
Visit 2 and 4. Urine will be collected throughout the Visit (over ~ 8 hours), for measurement of 
urine BHB. Urine pregnancy test will be also performed in women with child-bearing potential. 
See section 8.1.
Venous blood draws during Visit 5 
Up to 10 ml of blood will be collected for DNA collection to genotype subjects for the 
Apolipoprotein E (APOE) (the most significant genetic risk factor for AD) and the solute carrier 
family 16 member 7 (SLC16A7) genes; and potentially polymorphisms for other genes and other 
genetic factors.
Storage
The subject’s stored samples will be labeled with a code (such as letters and numbers) that only 
the study Investigators can link to the subject. Any identifying information about the subject will 
be kept confidential to the extent permitted by law. Samples acquired during this study will be 
tracked using the NIA Biological Sample Inventory system following NIH guidelines.
Samples of the subject’s blood and stool will be kept at the National Institutes on Aging, NIH Core 
Lab or one of our contract facilities; a subset of these samples may be retained at the Laboratory 
of Clinical Investigation (LCI) at the PI’s freezers. The subjects’ research samples will be stored 
with a confidential code and will be processed in batches at later times (not in real times). If consent 
for sharing was obtained in the original consent form, samples may be released to other physicians 
and scientists who are not listed as collaborators. The PI on this protocol will decide which co-
investigators and collaborating researchers may receive samples. The subjects’ samples may be 
used in their research only if the research has been approved by an Institutional Review Board 
(IRB). We will code the blood and stool samples and keep the subject’s identity confidential. We 
will retain a code list that enables us to link the clinical information and results on each sample to 
the subject.
We will code the blood that will keep the subject’s identity confidential. We will retain a code list 
that enables us to link the clinical information and results on each sample to the subject. DNA 
extraction from peripheral blood mononuclear cells (PBMCs) will take place for APOE and 
SLC16A7 genotyping and, potentially, additional genetic factors. We will not conduct Whole 
Genome (WGS) or Whole Exome Sequencing (WES). DNA samples will be stored at the NIA 
Core Lab. 
Access to research samples will be limited using either a locked room or a locked freezer. Samples 
and data will be stored using codes assigned by the investigators or their assigned designee(s). 
Data will be kept in password-protected computers. Only investigators or their designee(s) will 
have access to the samples and data.
At the Study Termination (after data analysis), samples and data will, after IRB approval, be 
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Data and sample sharing plan
Data and samples may be shared with collaborating laboratories at NIH or outside of NIH if 
consent for sharing was obtained in the original consent form. Samples/data that we may share in 
the future include biological specimens (blood and stool), MRI images and cognitive test results. 
If we wish to share samples in the future, 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) after prospective IRB approval. Required approvals from the 
collaborating institution will be obtained and materials will be shipped in accordance with NIH 
and federal regulations. No NIH or external collaborators that may receive samples have been 
identified at this time. 
8.2.6
Correlative Studies for Research
BHB quantification
BHB measurements will be conducted by ELISA.
EV biomarkers
Our Lab has been deriving blood EVs and enriching them for neuronal and astrocytic origin by 
means of immunocapture with antibodies against neuronal L1 Cell Adhesion Molecule and 
GLAST respectively and using them as a source of biomarkers for neurological disorders, such as 
AD and PD. We have shown that neuronal and astrocytic EV-based biomarkers reflect AD 
pathogenic cascades 63, insulin resistance 63,64, synaptic integrity 65, and neuronal homeostatic and 
metabolic processes 66.  Importantly, we have shown that EV-based biomarkers may be used in 
clinical trials to show target engagement and the level of the neuron and correlate with clinical 
outcomes (studies of experimental medications in AD 45, Parkinson’s disease 46, and dietary 
intervention in cancer 67). The proposed study will be the first using neuronal-enriched and 
astrocytic-enriched EVs to assess the effects of the KE drink supplementation compared to 
baseline and placebo. 
Specifically, we will assess EV biomarkers reflecting the following pathways of 
neuronal/astrocytic metabolism and AD pathogenesis: 
•
Insulin signaling and resistance: we will assess cascade in terms of total levels and 
activating phosphorylations at the level of IR, IRS-1, IGF1R, canonical cascade (Akt, 
GSK3B, S6K), alternative/mitogenic cascade (MAPK, JNK, p38), and mTOR/p-mTOR 
reflecting cellular autophagy (as in 45,46). We hypothesize that KE drink supplementation 
compared to baseline and placebo will improve overall cellular metabolism, thereby 
decreasing pSer-IRS1 phosphorylation characterizing insulin resistance, and will show 
activation of the overall cascade, which exerts neurotrophic effects.
•
Recently, we have shown that monocarboxylic acid transporters 1 and 2 (MCT-1 and 2), 
which import ketones into the cell, are present on neuronal and astrocytic exosomes. We 
hypothesize that KE drink supplementation compared to baseline and placebo will enhance 
neuronal ketone metabolism and may, therefore, upregulate the expression of relevant 
receptors and enzymes; this hypothesized upregulation may be reflected on circulating 

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exosomes. Therefore, we hypothesize that exosome levels of MCT-1, MCT-2, and other 
enzymes facilitating ketone metabolism will be higher after 28 days of KE drink compared 
to baseline and 28 days of isocaloric placebo supplement. 
•
Elevation of NAD+/NADH ratio has been proposed to mediate at least some of the 
beneficial effects of ketogenic interventions in neurodegeneration potentially through the 
effects on genes implicated in longevity and cellular health pathways such as the sirtuins 
and PARPs (Poly [ADP-ribose] polymerases) 68-70. Evidence shows that the NAD+/NADH 
ratio increases acutely (within hours in brain) 71. In addition, we have recently established 
that NAD+ and metabolites may be detected and quantified in plasma NEVs. Thus, we will 
test the hypothesis that KE drink supplementation compared to baseline and placebo will 
increase the NAD+/NADH ratio.
•
AD pathogenic cascades: we will measure A40, A42, p181-tau and total tau, which 
reflect the classic AD pathogenic cascades; complement and other neuroinflammatory 
mediators; other markers for diverse AD pathogenic processes (such as lysosomal 
dysfunction, cellular stress responses, and other factors that may be identified in the future) 
to assess potential alleviating/mitigating effects from  KE drink supplementation compared 
to baseline and placebo. Such evidence could be critical for motivating a potential future 
clinical trial of the KE drinks in AD.
•
A portion of EV preparations will be used for future measurements of RNA species 
involved in AD pathogenesis.
Stool microbiome
Stool samples will be subjected to microbiome analysis by RNA sequencing to determine changes 
in bacterial species composition between and within groups.
Test/assay
Volume blood 
(approximately)
Type of 
tube
Collection 
point (+/- 
48hrs)
Location of 
specimen 
analysis
EV isolates (fasting/before 
supplement):
•
Immunoassays (Western 
Blot, ELISA, 
electrochemiluminescence 
or similar)
•
RNA studies by qPCR
BHB (before and after 
supplement intake)
Visit 2: 40 ml 
(35 ml before/ 5 
ml after 
supplement)
Visit 3: 40 ml 
(35 ml before/ 5 
ml after 
supplement)
Visit 4: 55 ml 
(50 ml before/ 5 
ml after 
supplement)
EDTA 
(EVs)
Serum
(BHB)
Day 1 
(Visit 2), 
Day 14 ± 3 
(Visit 3), 
Day 28 ± 3 
(Visit 4)
Laboratory 
of Clinical 
Investigation 
(LCI) at 
Biomedical 
Research 
Center 
(BRC)/NIA

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Test/assay
Volume blood 
(approximately)
Type of 
tube
Collection 
point (+/- 
48hrs)
Location of 
specimen 
analysis
DNA collection 
Visit 5: 8ml 
PAXgene  Visit 5 
Laboratory 
of Clinical 
Investigation 
(LCI) at 
Biomedical 
Research 
Center 
(BRC)/NIA
8.2.7
Samples for Genetic/Genomic Analysis 
8.2.7.1   Description of the scope of genetic/genomic analyses
The DNA analysis conducted as part of this study is for research purposes only. Neither WGS nor 
WES is planned as part of the study.
Genetic testing can provide information about heritability of diseases and risk factors. This is 
particularly relevant for the ε4 allele of APOE gene, which increases the risk of Alzheimer’s 
disease and cardiovascular disease. However, carrying the ε4 allele does not guarantee that an 
individual will develop Alzheimer’s disease or coronary artery disease. 
8.2.7.2    Description of how privacy and confidentiality of medical information/biological 
specimens will be maximized
Blood will be used to extract DNA using standard laboratory techniques. The blood samples will 
be used for research purposes and never for medical genetic testing to define various types of 
genetic diseases. Participants’ genetic information will be kept confidential to the extent possible. 
The results will be kept in a locked and secured manner at the NIH. Genetic information will not 
be part of participants’ medical record and will not be revealed to others, including their relatives 
or doctor. Participants will not receive the results of any laboratory investigations involving the 
use of samples for genetic testing. 
8.2.7.3    Management of Primary Results
It is our policy to not disclose the results of genetic testing. 
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 clinically certified. 
In many cases, additional research may be necessary to determine whether these results are 
meaningful in terms of health and disease.  This is the case of the testing for the APOE gene that 
will take place in this study. In addition, testing for the APOE gene will take place after the 
participants’ study participation has ended and will not be immediately available for review. 

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8.2.7.4   Return of Secondary Genomic Research Results
N/A
8.2.7.5   Genetic counseling
N/A
8.3
SAFETY AND OTHER ASSESSMENTS
Physical Examination: This will be performed at the screening and final visits (Visits 1 and 4) by 
a study physician and will involve a general clinical examination focused on assessing eligibility 
criteria and neurological/psychiatric conditions and whenever indicated by the needs of the 
participants during the rest of the Visits (e.g. as clinically indicated, in case of reported side effect).
Nursing Examination: A nurse or certified nursing assistant will assess participants’ vital signs 
(temperature, Blood Pressure, Heart Rate, Respiratory Rate, Oxygen Saturation) at every visit and 
anthropometric measurements (height, weight, BMI, waist circumference) at the screening and 
final visits (Visits 1 and 4). *Height to be measured during visit 1 only.
Compliance Assessments: In addition to including a dedicated compliance visit (Visit 3), we will 
communicate with participants via phone call, once between Visit 2, 3 and once between Visits 3, 
4. Participants will also be advised to keep a log of the times of taking each dose. They will be 
asked to bring the Log in for review on Visits 3 and 4.
Credibility expectancy questionnaire (CEQ): This brief questionnaire will be administered on 
Visits 3 and 4 to assess expectations about effects of the supplement intake.
Neuropsychological Assessments: Cognitive testing will be administered by a study investigator 
during Screening, Visit 2 and Visit 4. The administered tests during screening will be CDR and 
MoCA. The administered tests during Visits 2 and 4 will be PACC and NIH Toolbox.
Clinical Labs (Visit 4): These will be performed at the screening and final visit (Visits 1 and 4) 
and include CBC with differential, Comprehensive Metabolic Panel including fasting glucose, 
Lipids panel (TGs, total cholesterol, LDL, HDL), HbA1c (will be performed by the NIA Harbor 
Lab), and fasting insulin (will be conducted by the Medstar Harbor Hospital lab).
Blood and urine BHB measurement: Blood will be collected at Visits 2, 3 and 4 for measurement 
of BHB and EV-biomarkers. Urine will be collected during Visits 2 and 4, for measurement of 
BHB. Blood will be drawn at the follow-up visit for DNA collection.
Magnetic Resonance Spectroscopy (MRS): Brain MRI/MRS and thigh muscle MRS will be 
performed at Visits 2 and 4. Muscle MRS will be optional.
Stool collection: Stool will be collected at Visits 2 and 4 for the assessment of intestinal 
microbiome.
General measures to monitor safety:
The investigator or clinical staff performing study procedure will monitor subject safety. This 
includes physicians and RNs during blood draws, nursing assessments and History and Physical 
sessions, investigators administering cognitive testing, MRI techs and investigators during MRIs. 

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Safety will be assessed with nursing assessments, History and Physical sessions, clinical labs, and 
by report of symptoms during phone calls or Visits.
Participants will be informed about new information from this or other studies that may affect their 
health, welfare, or willingness to stay in this study. Participants may decide to stop participating 
at any time. Any data and blood/stool collected until that point in time will remain part of the study 
and are the property of the National Institute on Aging. 
8.4
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS
8.4.1
Definition of Adverse Event
Adverse event means any untoward medical occurrence associated with the use of an intervention 
in humans, whether or not considered intervention-related (21 CFR 312.32 (a)).
8.4.2
Definition of Serious Adverse Events (SAE)
An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the view of the 
PI, it results in any of the following outcomes: death, a life-threatening adverse event, inpatient 
hospitalization or prolongation of existing hospitalization, a persistent or significant incapacity or 
substantial disruption of the ability to conduct normal life functions, or a congenital anomaly/birth 
defect. Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, they 
may jeopardize the participant and may require medical or surgical intervention to prevent one of 
the outcomes listed in this definition. Examples of such medical events include allergic 
bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias 
or convulsions that do not result in inpatient hospitalization, or the development of drug 
dependency or drug abuse.
8.4.3
Classification of an Adverse Event
Severity of Event
All AEs will be assessed by the PI according to the following grading system, which describes 
severity. 
•
Grade 1 Mild – Events require minimal or no treatment and do not interfere with the 
participant’s daily activities (e.g. mild nausea or bloating, unusually loose stools). 
•
Grade 2 Moderate – Events result in a low level of inconvenience or concern with the 
therapeutic measures. Moderate events may cause some interference with functioning (e.g. 
more than mild nausea that persists for several hours or results in vomiting; diarrhea).
•
Grade 3 Severe – Events interrupt a participant’s usual daily activity and may require 
systemic drug therapy or other treatment. Severe events are usually potentially life-
threatening or incapacitating (e.g. severe nausea with repeated vomiting or diarrhea 
resulting in orthostatic hypotension).  Of note, the term “severe” does not necessarily 
equate to “serious”. 
•
Grade 4 Severe: Life threatening or disabling AE

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•
Grade 5 Death
Relationship to Study Intervention
All adverse events (AEs) will have their relationship to study intervention assessed by the PI based 
on temporal relationship and his clinical judgment. The degree of certainty about causality will be 
graded using the categories below. The PI will keep in mind that in a clinical trial, the study product 
must always be suspect. 
•
Definitely Related – There is clear evidence to suggest a causal relationship, and other 
possible contributing factors can be ruled out. The clinical event, including an abnormal 
laboratory test result, occurs in a plausible time relationship to study intervention 
administration and cannot be explained by concurrent disease or other drugs or chemicals. 
The response to withdrawal of the study intervention (dechallenge) should be clinically 
plausible. The event must be pharmacologically or phenomenologically definitive, with 
use of a satisfactory rechallenge procedure if necessary.
•
Probably Related – There is evidence to suggest a causal relationship, and the influence 
of other factors is unlikely. The clinical event, including an abnormal laboratory test result, 
occurs within a reasonable time after administration of the study intervention, is unlikely 
to be attributed to concurrent disease or other drugs or chemicals, and follows a clinically 
reasonable response on withdrawal (dechallenge). Rechallenge information is not required 
to fulfill this definition.
•
Potentially Related – There is some evidence to suggest a causal relationship (e.g., the 
event occurred within a reasonable time after administration of the trial medication). 
However, other factors may have contributed to the event (e.g., the participant’s clinical 
condition, other concomitant events). Although an AE may rate only as “possibly related” 
soon after discovery, it can be flagged as requiring more information and later be upgraded 
to “probably related” or “definitely related”, as appropriate.
•
Unlikely to be related – A clinical event, including an abnormal laboratory test result, 
whose temporal relationship to study intervention administration makes a causal 
relationship improbable (e.g., the event did not occur within a reasonable time after 
administration of the study intervention) and in which other drugs or chemicals or 
underlying disease provides plausible explanations (e.g., the participant’s clinical 
condition, other concomitant treatments).
•
Not Related – The AE is completely independent of study intervention administration, 
and/or evidence exists that the event is definitely related to another etiology. There must 
be an alternative, definitive etiology documented by the clinician.
Expectedness 
The PI will be responsible for determining whether an adverse event (AE) is expected or 
unexpected.  An AE will be considered unexpected if the nature, severity, or frequency of the 
event is not consistent with the risk information previously described for the study intervention.

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8.4.4
Time Period and Frequency for Event Assessment and Follow-Up
The PI will make determinations regarding the presence and grading of on adverse events (AEs) 
and unanticipated problems (UPs). Whenever study investigators contact subjects throughout the 
study, they will seek information on AEs and UPs. AEs and UPs will also be elicited during study 
visits. Participants will be asked to use secure electronic mail or telephone to inform the 
investigators about any AEs or UPs experienced. AEs and UPs may be observed by the Investigator 
and/or study staff, elicited from the subject and/or family member, or volunteered by the study 
subject. AEs and UPs that had previously been reported by study subject will also be reassessed 
for duration, intensity and possible reoccurrence. Assessment of safety will include vital signs, 
history and physical sessions, questionnaires on psychological symptoms and clinical labs as 
indicated. The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to 
the attention upon review by a study monitor (Clinical Director or designee).
Any AE or UP that occurs between the times a study participant signs the informed consent form 
and the end of participation will be assessed and recorded under NIH Office of Human Subjects 
Research Protection policy #801. All AEs including local and systemic reactions not meeting the 
criteria for SAEs will be captured on the appropriate adverse event log. Information to be collected 
includes event description, time of onset, clinician’s assessment of severity, relationship to study 
product (assessed only by those with the training and authority to make a diagnosis), and time of 
resolution/stabilization of the event. All AEs occurring while on study must be documented 
appropriately regardless of relationship. All AEs will be followed to adequate resolution.
Any medical condition that is present at the time that the participant is screened will be considered 
past medical history and not reported as an AE. However, if the study participant’s condition 
deteriorates at any time during the study, it will be recorded as an AE. 
Changes in the severity of an AE will be documented to allow an assessment of the duration of the 
event at each level of severity to be performed. AEs characterized as intermittent require 
documentation of onset and duration of each episode.
The study team will record all reportable events with start dates occurring any time after informed 
consent is obtained until the last day of study participation.  At each study visit, the CRC and 
investigators will inquire about the occurrence of AE/SAEs since the last visit.  Events will be 
followed for outcome information until resolution or stabilization.
8.4.5
Adverse Event Reporting 
Adverse events, non-compliance both serious or continuing, protocol deviations both major and 
minor, as well as unanticipated problems are defined and described by the NIH Office of Human 
Subjects Research Protection policy #801 and will be reported in accordance with this policy. 
8.4.6
Serious Adverse Event Reporting 
Serious Adverse Events are defined and described by the NIH Office of Human Subjects 
Research Protection policy #801 and will be reported in accordance with this policy. 

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8.4.7
Reporting of Pregnancy 
Women with positive pregnancy test at screening will be excluded from the study (according to 
the exclusion criteria). If a participant becomes pregnant during the study, she will be discontinued 
from the study while being contacted until the end of the study to ensure safety. 
8.5
UNANTICIPATED PROBLEMS
8.5.1
Definition of Unanticipated Problems (UP)
Any incident, experience, or outcome that meets all of the following criteria:
•
Unexpected in terms of nature, severity, or frequency given (a) the research procedures 
that are described in the protocol-related documents, such as the Institutional Review 
Board (IRB)-approved research protocol and informed consent document; and (b) the 
characteristics of the participant population being studied; and
•
Related or possibly related to participation in the research (“possibly related” means there 
is a reasonable possibility that the incident, experience, or outcome may have been 
caused by the procedures involved in the research); and
•
Suggests that the research places participants or others (which many include research 
staff, family members or other individuals not directly participating in the research) at a 
greater risk of harm (including physical, psychological, economic, or social harm) than 
was previously known or expected.
8.5.2
 Unanticipated Problem Reporting 
The investigator will report unanticipated problems (UPs) to the NIH Institutional Review Board 
(IRB) as per Policy 801.
9
STATISTICAL CONSIDERATIONS
9.1
STATISTICAL HYPOTHESIS
Null Hypothesis (Ho): 
The administration of a KE drink in cognitively intact individuals of age ≥ 55 and MetS, will not 
increase brain BHB measured with MRS (primary endpoint) compared to baseline (within-subjects 
effect) and placebo (between-subjects effect). 
In addition, in the same population:
(i)
Ketone Ester drinks will not induce changes in EV biomarkers related to brain insulin 
resistance, autophagy, brain ketone metabolism, and AD pathology to a favorable 
direction (exploratory endpoint)
(ii)
Ketone Ester drinks will not improve performance on cognitive testing (exploratory 
endpoint)
Alternative Hypothesis (Ha):

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The administration of KE drinks in cognitively intact individuals of age ≥ 55 and MetS, will 
increase brain BHB measured with MRS (primary endpoint) compared to baseline (within-subjects 
effect) and placebo (between-subjects effect).
Also, in the same population:  
(i)
Ketone Ester drinks will induce changes in EV biomarkers related to brain insulin 
resistance, autophagy, brain ketone metabolism, and AD pathology to a favorable 
direction (exploratory endpoint)
(ii)
Ketone Ester drinks will improve performance on cognitive testing (exploratory 
endpoint)
 
9.2
SAMPLE SIZE DETERMINATION
The sample size in this study (n = 50 for complete data, 25 receiving KE drinks and 25 receiving 
placebo) gives the ability to detect an effect size (d) of 0.80 at a two-tailed Alpha of 0.05 and a 
Power of .80. In other concurrent studies, our ketone-sensitive PRESS sequence is capable of 
detecting an effect size (d) 0.72, roughly equivalent to an 18% change in BHB to creatine ratio. 
Since we know that brain ketone uptake is proportional to plasma ketones 15, we expect that brain 
BHB levels will change comparably to plasma BHB levels. A prior study using plasma 
measurements following KE administration versus placebo showed an effect size (d) of 4.05 32, 
suggesting more than adequate power for detecting brain BHB changes.   
Also, in a previous study (Evans et al) 72, 8 healthy young male athletes were tested on a domain 
of cognitive performance (executive function) before and after a period during which they were 
taking a ketone supplement (similar but not identical to the KE of the present study) while 
exercising 72. This intervention resulted in peripheral BHB of ~1.5 to 2.6 mM (less than the level 
of peripheral BHB elevation we expect with the KE) and showed a significantly better executive 
function performance, with an effect size (d) of 0.7 72. Since in our proposed study we expect to 
achieve a greater peripheral BHB induction and our sample size is much greater than Evans et al 
72, it is likely that our study is powered well enough to detect cognitive changes.
9.3
POPULATIONS FOR ANALYSES
A “Modified Intention-to-Treat Analysis” will be performed including participants that took at 
least one dose of the intervention and excluding those who never took the intervention. Finally, 
we will perform a “Per-Protocol Analysis” including those participants who took the intervention 
for at least for 80% of days of the study’s duration based on compliance logs. 
9.3.1
Evaluable for toxicity 
All patients will be evaluable for toxicity from the time of their first treatment with KE drinks or 
placebo. 
9.3.2
Evaluable for objective response 
Only those patients who have had measured levels of 
(1) Serum BHB 
(2) MRS BHB, 

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(3) EV biomarkers
(4) Cognitive outcomes
at baseline and at least once after the intervention, will be considered evaluable for response. 
9.4
STATISTICAL ANALYSES
9.4.1
General Approach
First, we will test the distribution pattern of our data (normal vs non-normal). Continuous data will 
be presented as means (standard deviations) in case of normal distribution and as medians (ranges) 
for non-normal distributions. Categorical data will be presented as percentages. Parametric or non-
parametric statistical tests will be used based on the distribution of the data (normal, non-normal). 
In case which it is clinically meaningful, we will transform continuous data into categorical data 
by creating clinically meaningful categories of the continuous data. 
Criteria for statistical significance will be set at alpha of 0.05 (two-tailed tests). Potential covariates 
that will be considered in the statistical analyses are age, sex, ethnicity, BMI, waist circumference, 
TGs, glucose levels, HDL levels, Blood Pressure levels, baseline cognitive status. 
9.4.2
Analysis of the Primary Endpoints
The primary endpoint will be continuous and will be presented as mean (standard deviation).  It 
will be measured as MRS BHB concentration normalized to Creatine (BHB/Creatine ratio) in 
bilateral precuneus. For the primary endpoint, repeated-measures mixed models analysis will be 
performed to examine the effects of the between-subjects factor “Group” (KE drink vs. Placebo 
group), the within-subjects factor “Time” and their Interaction, separately for acute and chronic 
effects (Baseline vs. Outcome). Exploratory analyses will include age, sex, weight/BMI, waist 
circumference, TGs, glucose levels, HDL levels, Blood Pressure levels (reflecting severity of 
MetS), cognitive status, peripheral ketone levels and EV biomarkers, entered as covariates in the 
mixed model. If a covariate is not substantively related to the model (p > 0.15), it will be dropped. 
9.4.3
Analysis of the Secondary Endpoint(s)
To assess whether genetic factors modulate the response to the KE supplement, in the repeated-
measures mixed models, we will include the APOE ε4 carrier status (ε4 carriers, ε4 non-carriers), 
the SLC16A7, and potentially other polymorphisms, as additional fixed effect factors and their 
interactions with “Group” and “Time”.
9.4.4
Safety Analyses
The study will not evaluate safety endpoints formally. However, AEs will be published at the time 
of the final research report and will be analyzed as summary statistics. We will report AEs as 
incidence per administration of total doses of KE drink vs Placebo. Also, we will report AEs as 
number of participants with a specific AE per group. In case of an AE presenting with different 
severities, we will subcategorize incidence based on the severity group. 

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9.4.5
Baseline Descriptive Statistics
We will present baseline characteristics including demographics, clinical labs, and anthropometric 
characteristics (age, sex, ethnicity, BMI, weight, waist circumference, TGs, glucose levels, HDL 
levels, Blood Pressure levels, cognitive status, peripheral and brain ketone levels (BHB, AcAc), 
EV biomarker levels) using descriptive statistics. Inferential statistics will not be used. 
9.4.6
Planned Interim Analysis
N/A
9.4.7
Sub-Group Analyses
We may perform exploratory subgroup analyses based on age groups, sex, ethnicity, baseline 
cognitive status or other factors, especially if there are baseline differences. We may also perform 
exploratory sensitivity analyses using the responses of the credibility expectancy questionnaire as 
covariate to assess any effects of subjective expectations.
9.4.8
Tabulation of individual Participant Data
Individual participant data will be listed by measure and time point. 
9.4.9
Exploratory Analyses
All analyses related to serum BHB, EV biomarkers, cognitive performance, thigh MRS and stool 
microbiome will be exploratory. Also, exploratory analyses will be performed for the primary 
endpoint to include additional covariates to the mixed model.
10 REGULATORY AND OPERATIONAL CONSIDERATIONS
10.1 INFORMED CONSENT PROCESS
10.1.1 Consent/Assent Procedures and Documentation
The consent will be obtained in person at the NIA Clinical Unit. The PI, clinical research 
coordinator (nurse), associate investigators, and NIA research nurses and nurse practitioners will 
be authorized to obtain consent. 
All participants will receive a verbal explanation in terms suited to their comprehension of the 
purposes, procedures and potential risk 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.   
When the participant fully understands her responsibilities and possible risks and discomforts and 
agrees to participate in the current study, they will be provided with the consent to sign. A signed 
copy will be provided to each participant, as well as placed in her medical record. No procedures 
will begin until the informed consent has been properly obtained.

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In particular, the summary study design will be detailed. Furthermore, participants will be fully 
informed about the data and safety monitoring procedures.
The informed consent procedure might last up to an hour according to our previous experience, 
allowing for thorough explanation of the study’s procedures and answer of possible questions. The 
clinical research coordinator (nurse) and the PI will be available to answer any question that 
participants may have. Participants can have additional time to think and decide on their 
participation. Additionally, they will have the option to discuss with their families and/or friends 
and/or primary physician before they provide consent.  
When an NIH staff member in our work unit seeks to enroll in the study, a consent monitor who 
is independent of the research team will be present to observe the consent process. Conversely, 
we will contact the CC Department of Bioethics Consultation Service or the NIMH Human 
Subjects Protection Unit (HSPU) in order to enlist a consent monitor who can observe remotely. 
If no such person is available, consent observation will be performed by another qualified 
investigator on the study other than the staff member's supervisor.
10.1.2 Participation of Subjects who are/become Decisionally Impaired
Adults unable to give consent are excluded from enrolling in the protocol. If during study, a 
previously eligible participant becomes decisionally impaired she/he will be withdrawn from the 
study.
10.2 STUDY DISCONTINUATION AND CLOSURE
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause.  If the study is prematurely terminated or suspended, the PI will promptly inform 
study participants and the IRB and will provide the reason(s) for the termination or suspension. 
Study participants will be contacted, as applicable, and be informed of changes to study visit 
schedule.
Circumstances that may warrant termination or suspension include, but are not limited to:
•
Determination of unexpected, significant, or unacceptable risk to participants
•
Insufficient compliance to protocol requirements
•
Data that are not sufficiently complete and/or evaluable
Study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the IRB and/or Food and Drug Administration (FDA).
10.3 CONFIDENTIALITY AND PRIVACY
Participant confidentiality and privacy is strictly held in trust by the participating investigators and 
their staff. This confidentiality is extended to cover testing of biological samples in addition to the 
clinical information relating to participants. Therefore, the study protocol, documentation, data, 
and all other information generated will be held in strict confidence. 
All research activities will be conducted in as private a setting as possible.

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Identifiers will be attached to medical records and to data stored in password-protected computers 
and servers but not to samples. All samples will be coded and the link of these codes will be 
available to the PI and other investigators. Access to the “key” for the code will be limited to 
investigators in this study. Data or samples may be shared with other researchers at the PI’s lab 
and other NIH investigators to conduct biomarker measurements, but the “key” will not be shared 
with them. Personally identifiable information will not be released to third parties, unless required 
by law (specifically, HIV, HBV, HCV results).
The study monitor (NIA Clinical Director or designee), representatives of the Institutional Review 
Board (IRB), and/or regulatory agencies may inspect all documents and records required to be 
maintained by the investigator, including but not limited to, medical records (office, clinic, or 
hospital) and pharmacy records for the participants in this study. The clinical study site will permit 
access to such records.
The study participant’s contact information will be securely stored at each clinical site for internal 
use during the study. At the end of the study, all records will continue to be kept in a secure location 
for as long a period as dictated by the reviewing IRB or Institutional policies requirements.
Study participant research data, which is for purposes of statistical analysis and scientific reporting, 
will be transmitted to and stored at NIA-approved servers and software platforms that grant limited 
access to study PI and designees and are secure and password-protected. The participant’s contact 
or identifying information will only be stored in the medical record and NIA-approved clinical 
application electronic platform. In servers containing research data, individual participants and 
their research data will be identified by a unique study identification number. 
To further protect the privacy of study participants, a Certificate of Confidentiality has been issued 
by the National Institutes of Health (NIH).  This certificate protects identifiable research 
information from forced disclosure. It allows the investigator and others who have access to 
research records to refuse to disclose identifying information on research participation in any civil, 
criminal, administrative, legislative, or other proceeding, whether at the federal, state, or local 
level. By protecting researchers and institutions from being compelled to disclose information that 
would identify research participants, Certificates of Confidentiality help achieve the research 
objectives and promote participation in studies by helping assure confidentiality and privacy to 
participants.
10.4 FUTURE USE OF STORED SPECIMENS AND DATA
Data and samples may be shared with collaborating laboratories at the NIH or outside of the NIH 
if consent for sharing was obtained in the original consent form. Samples/data that we may share 
in the future include biological specimens (blood/DNA/stool), MRI images, MRS data, cognitive 
test results, and biomarker data.
Samples and data collected under this protocol may be used to study physiology related to brain 
function and metabolism. The subject’s stored samples will be labeled with a code (such as letters 
and numbers) that only the study team can link to the subject. Any identifying information about 
the subject will be kept confidential to the extent permitted by law. Samples acquired during this 
study will be tracked using the NIA Biological Sample Inventory system following NIH 
guidelines.  

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Samples of the subject’s blood will be kept at the Lab of the PI at the National Institute on Aging 
Biomedical Research Center, the NIA Core Lab or one of our contract facilities. The subject’s 
samples may be tested immediately, or they may be frozen and used later. The subject’s samples 
will be stored with a confidential code. Samples may be kept until no cells remain or until the 
investigators decide to destroy them. If the subject gives us permission some samples may be 
released to other doctors and scientists who are not associated with this institute. The Principal 
Investigator on this protocol will decide which co-investigators and collaborating researchers may 
receive samples. The subject’s samples may be used in their research only if the research has been 
approved by an Institutional Review Board (IRB) and is related to the original research questions 
association with this protocol or for other research purposes as indicated below
We will code the blood that will keep the subject’s identity confidential. We will retain a code list 
that enables us to link the clinical information and results on each sample to the subject
Access to research will be limited using either a locked room or a locked freezer. Samples and data 
will be stored using codes assigned by the investigators or their assigned designee(s). Data will be 
kept password-protected computers. Only investigators or their designee(s) will have access to the 
samples and data.
At the completion of the protocol (termination), samples and data will either be destroyed, or after 
IRB approval, transferred to another existing protocol or a repository protocol.
10.5 SAFETY OVERSIGHT
Safety oversight will be under the direction of the NIA Clinical Director or designee, who will 
assess safety data. A report will be provided to the IRB at the time of the continuing review.
10.6 CLINICAL MONITORING
Clinical monitoring is conducted to ensure that the rights and well-being of trial participants are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct 
of the trial is in compliance with the currently approved protocol/amendment(s), with International 
Conference on Harmonisation Good Clinical Practice (ICH GCP), and with applicable regulatory 
requirement(s). 
The PI will provide data and safety monitoring. In addition, an independent data and safety 
monitor (NIA Clinical Director or designee) will review every 6 months any AEs, SAEs and UPs 
and determine the need for suspension of enrollment or other action. In addition, this protocol 
will undergo a review by the NIA Clinical Investigator Committee every year, which is standard 
for all NIA clinical protocols.
10.7 QUALITY ASSURANCE AND QUALITY CONTROL
The PI will be responsible for Quality Assurance for this study. The PI will address any issues 
discovered. The PI or designee investigator will perform internal quality management of study 
conduct, data and biological specimen collection, documentation and completion.  Quality control 
(QC) procedures will be implemented beginning with the data entry system and data QC checks 

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that will be run on the database will be generated. Any missing data or data anomalies will be 
communicated to the PI for clarification/resolution.
10.8 DATA HANDLING AND RECORD KEEPING
10.8.1 Data Collection and Management Responsibilities
We will retain study documents for a minimum of 2 years after the publication of the results or 3 
years after we acquire all data needed for the analysis.
10.9 PROTOCOL DEVIATIONS
It is the responsibility of the PI to use continuous vigilance to identify and report deviations to the 
NIH Institutional Review Board as per Policy 801. All deviations must be addressed in study 
source documents, reported to NIA Program Official.  The PI is responsible for knowing and 
adhering to the reviewing IRB requirements.
10.9.1 NIH Definition of Protocol Deviation
A protocol deviation is any changed, divergence, or departure from the IRB-approved research 
protocol. 
•
Major deviations: Deviations from the IRB approved protocol that have, or may have the 
potential to, negatively impact the rights, welfare or safety of the subject, or to 
substantially negatively impact the scientific integrity or validity of the study.
•
Minor deviations: Deviations that do not have the potential to negatively impact the 
rights, safety or welfare of subjects or others, or the scientific integrity or validity of the 
study.
10.10   PUBLICATION AND DATA SHARING POLICY
10.10.1  Human Data Sharing Plan
This study will be conducted in accordance with the following publication and data sharing 
policies and regulations:
National Institutes of Health (NIH) Public Access Policy, which ensures that the public has access 
to the published results of NIH funded research. It requires scientists to submit final peer-reviewed 
journal manuscripts that arise from NIH funds to the digital archive PubMed Central upon 
acceptance for publication.
This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of NIH-
Funded Clinical Trial Information and the Clinical Trials Registration and Results Information 
Submission rule. As such, this trial will be registered at ClinicalTrials.gov, and results information 
from this trial will be submitted to ClinicalTrials.gov. In addition, every attempt will be made to 
publish results in peer-reviewed journals.  Data from this study may be requested from other 
researchers 2 years after the completion of the primary endpoint or any time after results of the 
study have been accepted for publication in a peer-reviewed journal, by contacting the PI.

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10.11  CONFLICT OF INTEREST POLICY
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical.  Therefore, any actual conflict of interest of persons who have 
a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and 
managed. Furthermore, persons who have a perceived conflict of interest will be required to have 
such conflicts managed in a way that is appropriate to their participation in the design and conduct 
of this trial.  The study leadership in conjunction with the NIA has established policies and 
procedures for all study group members to disclose all conflicts of interest and will establish a 
mechanism for the management of all reported dualities of interest.
11 ABBREVIATIONS
AE
Adverse Event
ANCOVA
Analysis of Covariance
AcAc
Aceto-Acetate
APOE
Apolipoprotein E
BHB
β-hydroxybutyrate
CFR
Code of Federal Regulations
CLIA
Clinical Laboratory Improvement Amendments
CMP
Clinical Monitoring Plan
COC
Certificate of Confidentiality
CONSORT
Consolidated Standards of Reporting Trials
CRF
Case Report Form
DCC
Data Coordinating Center
DHHS
Department of Health and Human Services
DNA
Deoxyribonucleic acid
DSMB
Data Safety Monitoring Board
DRE
Disease-Related Event
DXT
Dextrose
EC
Ethics Committee
eCRF
Electronic Case Report Forms
FDA
Food and Drug Administration
FDAAA
Food and Drug Administration Amendments Act of 2007
FFR
Federal Financial Report
GCP
Good Clinical Practice
GLP
Good Laboratory Practices
GMP
Good Manufacturing Practices
GWAS
Genome-Wide Association Studies
HIPAA
Health Insurance Portability and Accountability Act 
IB
Investigator’s Brochure
ICH
International Conference on Harmonisation 
ICMJE
International Committee of Medical Journal Editors
IDE
Investigational Device Exemption

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IND
Investigational New Drug Application
IRB
Institutional Review Board
ISM
Independent Safety Monitor
ISO
International Organization for Standardization
ITT
Intention-To-Treat
KE
Ketone Ester
KS
Ketone Salt
LSMEANS
Least-squares Means
MedDRA
Medical Dictionary for Regulatory Activities
MOP
Manual of Procedures
MSDS
Material Safety Data Sheet
MRI
Magnetic Resonance Imaging
MRS
Magnetic Resonance Spectroscopy
NCT
National Clinical Trial
NIH 
National Institutes of Health
NIH IC
NIH Institute or Center
OHRP
Office for Human Research Protections
PI
Principal Investigator
QA
Quality Assurance
QC
Quality Control
SAE
Serious Adverse Event
SAP
Statistical Analysis Plan
SLC16A7
Solute Carrier Family 16 Member 7
SMC
Safety Monitoring Committee
SOA
Schedule of Activities
SOC
System Organ Class
SOP
Standard Operating Procedure
UP
Unanticipated Problem
US
United States
WES
Whole Exome Sequencing
WGS
Whole Genome Sequencing
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