Strategies to Augment Ketosis: Optimization of Ketone Delivery Strategies
Phase: NA
Conditions: Ketosis
Sponsor: Ohio State University

Strategies to Augment Ketosis: 
Optimization of Ketone 
Delivery(STAK:OK’d) 
 
The Ohio State University 
 
IRB Approval   July 26, 2022 
 
IRB# 2022H0160 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Strategies to Augment Ketosis: Optimization of Ketone Delivery (STAK: OK’d) IRB 
Protocol 
 
 
Title:  
Optimization of Ketone Delivery 
Short title: 
 
STAK-OK’d 
 
PI: 
 
 
Jeff S. Volek, PhD, RD 
Professor, Department of Human Sciences 
The Ohio State University 
305 Annie & John Glenn Ave 
Columbus, OH 43210 
Office: 614-688-1701 | volek.1@osu.edu 
 
Co-PI:  
 
Brianna Stubbs, DPhil 
 
 
 
Lead Translational Scientist  
The Buck Institue for Research on Aging 
8001 Redwood Blvd 
Novato, CA 94945 
Office: 415-209-2000 | BStubbs@buckinstitute.org  
 
 

ABBREVIATIONS 
AcAc 
Acetoacetate 
BHB 
Beta hydroxybutyrate 
KE 
Ketone ester 
BDO 
(R)-1,3 Butanediol 
BTQ 
Beverage tolerability questionnaire 
LBM 
Lean body mass 
LC-MS 
Liquid chromatography – mass spectrometry  
VO2 
Oxygen uptake 
VCO2 
Carbon dioxide release 
GRAS 
Generally recognized as safe 
AUC 
Area under the curve 
Cmax 
Maximal concentration 
Cmin 
Minimal concentration 
Tmax 
Time of maximal concentration 
Tmin 
Time of minimal concentration 
mL 
milliliters 
mg 
Milligrams 
h 
Hour 
ANOVA 
Analysis of variance 
AE 
Adverse event 
USG 
Urine Specific Gravity  
 
 
 

STUDY SYNOPSIS 
 
 
 
Study Title 
Optimization of Ketone Delivery 
Short Title 
STAK-OK’d 
Study Design 
Open label, randomized, placebo controlled, 9-arm cross over 
Study Participants 
• Healthy men, aged 20 -30 years old,  BMI between 18 
and 29 kg/m2 
Planned Sample Size 
An evaluable sample of 12 participants/group is required; to 
allow for 20% attrition a final sample size of 15 is planned.  
Study Products 
1. BHB Mono-ester 
2. C6 Ketone Di-ester 
3. AcAc Di-ester 
4. (R)-1,3 butanediol 
5. Control 
All study products are generally recognized as safe (GRAS) 
food ingredients.  
Serving Sizes 
1. 180 mg/kg of lean body mass 
2. 360 mg/kg of lean body mass 
Planned Study Period September 2022 – September 2023 
OUTCOMES 
Primary 
Difference in total plasma ketone appearance (AUC) between 
the two serving sizes of study products and control. 
Secondary 
• Differences in other blood ketone kinetic parameters (peak 
concentration, time of peak concentration, AUC of different 
ketone bodies, capillary ketone concentrations).  
• Differences in urine and breath ketone excretion. 
• Differences in blood metabolites (glucose, free fatty acids, 
alanine). 
• Differences in blood hormones (insulin, ghrelin). 
• Differences in blood acid-base balance (bicarbonate, strong 
ions). 
• Differences in gas exchange (VO2 and VCO2). 
• Differences in heart rate and heart rate variability and blood 
pressure. 
• Differences in tolerability and subjective satiety.  

SUMMARY AND BACKGROUND  
This project is focused on nutrition-based STRATEGIES TO AUGMENT KETOSIS (STAK), 
which will enable people to increase blood ketone concentrations, achieve ‘nutritional 
ketosis’ which may possibly have beneficial effects on resilience and health. Ketone esters 
(KEs) represent one promising STAK method, but even within this category of exogenous 
ketones there are multiple compounds that vary in composition of ketone-promoting 
components joined by ester bonds. The ketone-promoting components can be ketone 
bodies such as beta hydroxybutyrate (BHB) or acetoacetate (AcAc), ketogenic precursors 
that metabolize to ketones via the classical beta-oxidation ketogenesis pathway (i.e., 
medium chain fatty acids) or ketone precursors that metabolize to BHB via a non-classical 
pathway (i.e., (R) -1,3 butanediol, BDO). Three Generally Recognized as Safe (GRAS) KE 
compounds exist, each with distinct pharmacokinetic and pharmacodynamic effects. Most 
recent clinical KE research has used a monoester of BHB and (R)-1,3 butanediol (BHB 
Mono-ester), with two more recently available compounds including a diester of hexanoic 
acid (a ketogenic medium chain fatty acid) and (R)-1,3 butanediol (C6 Di-ester) and a 
diester of AcAc and (R,S)-1,3 butanediol (AcAc Di-ester)(Fig 1 and Table 1). Whilst blood 
BHB kinetics of the BHB Monoester are well characterized at rest and during exercise1-11, 
human studies of the two other KE compounds are limited. Recent studies of KEs suggest 
that they may have many acute functional effects, such as modulation of glucose, fatty acid 
and amino acid metabolism5,6, heart rate4, acid base homeostasis7,8,11,12 and urine output7,8, as 
well as multiple other effects demonstrated in preclinical research, including modulation of 
oxidative stress and inflammation13,14, that are yet to be investigated in clinical studies. As 
the number and sophistication of KE products grows, there is a critical need to expand our 
knowledge of the similarities and differences between these compounds and how their 
metabolism varies between individuals to optimize dosing strategies for service members . 
Our long- term goal is to create evidence-based guidelines for deployment of diverse KE 
based on knowledge of each compound, the effect of individual phenotype, and operational 
functional requirements.  
 
KE Metabolic Effects Vary Between Compounds. One important difference between KE 
compounds is the ketone-promoting components, which determines the circulating ratio of 
blood ketone bodies, BHB and AcAc, and may in turn lead to important metabolic and 
signaling differences. Whereas some actions of the ketone bodies BHB and AcAc are 
shared, R-BHB has a broad range of signaling functions that are distinct from AcAc, some 
of which are shared by the non-circulating, non-oxidizable enantiomer, S-BHB13,14. AcAc also 
has metabolic and signaling actions that are independent of BHB15-18 and is selectively 
oxidized in some cells that cannot oxidize BHB42. Furthermore, responses to different ketone 
bodies vary between tissue types15. A second difference between KE arises from the 
balance between direct delivery of ketones compared to indirectly elevating ketone 
concentration via metabolism of non-classical or classical ketogenic precursors (Figure 1). 
Classical ketogenesis itself may drive adaptation and some of the functional benefits 
associated with ketosis19. BDO is included in all of the KE compounds, but it is currently 

unknown how consumption of BDO alone, and its metabolism via non-classical ketogenesis 
acutely affects metabolism. Additionally, ketogenesis is now understood to occur in certain 
cells outside the liver with important local biological effects, for example ketogenesis driven 
by medium chain fatty acids has been reported in astrocytes in vitro10. Provision of systemic 
BHB by a KE may elicit different biological effects in some tissues such as the brain versus 
promoting in situ ketogenesis in that tissue. Overall, not only are functional effects of KE 
incompletely defined, but also it is unknown which effects are common to all KE versus 
which are specific to an individual KE compound (i.e., BHB Monoester vs AcAc Diester) or 
which may be attributable to the BDO precursor common to all of the KE.   
 
This study will be the first comparative full crossover study of all available KE and the 
precursor BDO at two serving sizes. Outcomes will focus on established effects of the BHB 
Monoester (including the effects on ketones, glucose and acid-base balance) and compare 
these with the effects of the AcAc Diester, C6 Diester and BDO. The results of this project 
will provide information to facilitate selection of exogenous ketones based on the specific 
physiological and metabolic effects that they induce.   
 
Figure 1| Metabolism of 3 ketone ester compounds and the BDO ketone-precursor. 

IMPACT   
KEs could represent a practical STAK method that does not require any change in diet. 
However, there is a critical need to elucidate unique attributes of the many new 
exogenous ketone compounds and how their metabolism varies between individuals to 
inform KE use in the field. This high impact project will contribute important knowledge 
that aims to develop next-generation KE molecules and formulations that are designed 
to meet the needs of operators at different points in their career. Our long-term goal is to 
create evidence-based guidelines for deployment of diverse KE compounds, dose level, 
and timing based on individual characteristics and operational/training requirements. 
 
RESEARCH STRATEGY  
This is an open label, randomized, placebo controlled, 9-arm crossover study where healthy 
adult men (n = 15) will consume four different exogenous ketone products at two serving 
sizes or a non-ketogenic placebo (one study product per test day). Data/sample collection 
will include blood (capillary and whole venous blood), gas exchange measures, urine, heart 
rate/variability and study product tolerability.   
 
OUTCOMES 
Primary  
• 
Difference in total plasma ketone appearance (AUC) between the two serving sizes 
of study products and control 
Secondary* 
• 
Differences in other blood ketone kinetic parameters (peak concentration, time of 
peak concentration, AUC of different ketone bodies, capillary ketone concentrations). 
• 
Differences in urine and breath ketone excretion  
• 
Differences in blood metabolites (glucose, free fatty acids, alanine). 
• 
Differences in heart rate and heart rate variability and blood pressure 
• 
Differences in subjective tolerability and subjective satiety measured by 
questionnaires 
• 
Differences in blood hormones (insulin, ghrelin). 
• 
Differences in blood acid-base balance (bicarbonate, strong ions 
• 
Differences in respiratory gas exchange (VO2 and VCO2 
*All comparisons will be made between the two serving sizes of one study product and vs. 
control.  
 
PARTICIPANTS 
This study aims to recruit a homogenous, military relevant population to remove the 
confounding effects of variation in phenotype on exogenous ketone metabolism as far 
as possible. Thus, we will 15 recruit healthy and normally-active resistance/endurance 
trained men from the ages of 20-30 yr that represents typical active-duty military 

personnel. Each participant must meet all of the inclusion criteria and none of the 
exclusion criteria at screening in order to participate 
 
Inclusion criteria: 
• Male 
• BMI between 18 and 29 kg/m2 
• Aged 20 – 30 years 
• Participant is willing and able to comply with all study procedures including the 
following prior to Test Days: fasting (>10 h; water only), no alcohol (>24 h), no  
exercise (>24 h), no acute illness and controlled feeding before each Test Day, 
maintain diet, exercise, medication, and supplement habits throughout the study.  
• Participant has no health conditions that would prevent completion of the study 
requirements as judged by the Investigator based on health history.  
• Participant understands the study procedures and signs forms providing informed 
consent to participate in the study and authorizes the release of relevant 
protected health information to the Investigator.  
Exclusion criteria: 
• Participant follows a low-carbohydrate diet (<30% energy from carbohydrate) or 
have used exogenous ketone supplements within 4-months of study 
participation. 
• Participant has a Primary Care Physician diagnosed history or presence of 
uncontrolled and/or clinically important hypertension (blood pressure >150/95 
mmHg), pulmonary, cardiac, hepatic, renal, endocrine (including type 1 and 2 
diabetes), hematologic, immunologic, neurologic (e.g., Alzheimer’s or 
Parkinson’s diseases), psychiatric (including unstable depression and/or anxiety 
disorders) or biliary disorders.  
• Participant has a known allergy, intolerance, or sensitivity to any of the 
ingredients in the study beverages, including soy and milk protein, wheat, 
shellfish, fin fish, eggs, tree nuts or peanuts (production facility handles nuts).  
• Participant has unstable use of a medication or supplement that the Investigator 
considers may affect the outcomes of the trial.  
• Consumption of alcohol more than 3 drinks per day or more than 18 drinks per 
week.  
• Consumption of tobacco. 
• Consumption of cannabis. 
• Participant is currently in another research study or has been in the 14 days 
before screening.  
• Participant has had a blood draw or donation in the last 8 weeks.  
• Participant has a clinically important gastrointestinal (GI) condition that would 
potentially interfere with the evaluation of the study beverage [e.g., inflammatory 

bowel disease, irritable bowel syndrome, chronic constipation, severe 
constipation (in the opinion of the Investigator), history of frequent diarrhea, 
history of surgery for weight loss, gastroparesis, systemic disease that might 
affect gut motility according to the Investigator, medication managed reflux 
and/or clinically important lactose intolerance].  
• Participant has a condition the Investigator believes would interfere with his 
ability to provide informed consent, comply with the study protocol, which might 
confound the interpretation of the study results, or put the participant at undue 
risk.  
 
Sample Size. Our primary outcome goal is to demonstrate significant differences between 
serving sizes of exogenous ketone compounds in plasma total ketone AUC. Previous 
work using the BHB Monoester compared to ketone salt products demonstrated that a 
sample size of 12 participants was required to detect a difference in BHB AUC of ~20% 
(80 mM.h-1) between ketone compounds with alpha set at 0.5 and power at 0.8. This 
sample size will also give sufficient power to address our secondary aims which include 
differences in peak ketone concentrations, plasma BHB, AcAc, breath acetone, glucose, 
insulin, free-fatty acids, amino acids, blood chemistry, pH, gas exchange values and heart 
rate. Based upon prior studies, we expect up to 20% of participants will withdraw from the 
study after recruitment, and therefore we will plan to recruit 15 participants.  
 
Recruitment: 
Recruitment will occur through well-established methods at OSU, which include word of 
mouth, ResearchMatch database, social media engagement and flyers. For this study 
we will specifically aim to recruit cadets in the OSU Reserve Officers Training Corps 
(ROTC), who have participated in research on ketosis in the past and been supported 
by their cadre to enroll in our research projects (see letters of support from Army, Navy, 
and Air Force ROTC leadership).   
  
Randomization: 
If a participant meets all inclusion and none of the exclusion criteria, the following will 
occur at the end of the screening visit:  
• The randomization sequence will be prepared using an online random sequence 
generator (randomization.com) that will generate a list of random number 
sequences between 1 – 9 that correspond to product/serving code. If a participant 
meets all inclusion and none of the exclusion, a staff member will randomize the 
participant – in the order that they are enrolled. The randomization number will be 
recorded in the participant's source documentation.  
 
METHODS  

Study overview:  
 
 Figure 2 | Study schematic, showing timeline for a study day.   
 
Study Procedures 
Study Day 
Screening 
Test Days 1-9 
Visit to Study Center 
X 
X 
Informed consent 
X 
 
Medical intake questionnaire, taste screening, 
diet assessment, anthropometrics1 
X 
 
Randomization2 
X 
 
Compliance assessment3 
 
X 
Consume study product4 
 
X 
IV Cannulation and whole blood collection5 
 
X 
Urine collection6 
 
X 
Respiratory gas analysis7 
 
X 
Heart rate and HRV monitoring8 
 
X 
Tolerability assessment9 
 
X 
Satiety assessment10 
 
X 
Continuous Ketone Monitor placement/check11 
 
X 
 
1. Medical intake questionnaire, taste screening diet assessment questionnaire and 
anthropometric data will include: Lean Body Mass by DXA, BMI, height and weight. 
2. Order of study products will be randomized using a computer randomization generator.  
3. Prior to starting each test day, you will be asked to confirm that you meet pre-test criteria, 
including: fasted >10h, no alcohol for 24h, no exercise for 24h and consumed the provided 
pre-test food.  

4. Study Product will be consumed within +/- 60 minutes of the time established on the first 
test day. Participants will be provided with a choice of non-caffeinated, non-caloric beverage 
to remove the bitter taste of the Study Product.   
5. IV cannula will be inserted at the start of each Test Day, and removed at the end of each 
Test Day, Blood samples will be collected according to the schedule in Figure 1. Cannula will 
be flushed with a small volume of saline after each sample to maintain patency. We will draw 
504 mL of blood, which is about 2.1 cups throughout the 4-week intervention.  
6. Prior to consumption of the Study Product, participants will be asked to completely void 
your bladder. And hydration status will be determined via urine specific gravity (USG) reporting 
<1.025.  Urine passed after the ingestion of the study product will be collected in a plastic 
container; you will be asked to void their bladder and collect urine at the end of the test day. 
The volume produced will be recorded at the end of the study and aliquots will be frozen and 
stored for future analysis. 
7. Participants will breathe into a commercially available handheld breath acetone analyzer 
according to the schedule in Figure 2. Participants will wear a fitted face mask attached to a 
metabolic cart for a 10-minute period every 60 minutes. 
8. Participants will wear a Bluetooth heart rate monitor chest strap throughout the test day.  
9. Participants will complete a Beverage Tolerability Questionnaire prior to Study Product 
consumption and at the end of the Test Day. 
10. Participants will complete a 3-item satiety visual analogue scale prior to Study Product 
consumption and at the end of the Test Day. 
11. Continuous Ketone Monitor will be applied at the start of Test Day 1. The sensor will be 
checked by the study team at each test day and will be removed and replaced by a fresh 
sensor at ~2- week intervals during the study. The sensor will be removed at the end of the 
final test day.  
 
Screening Visit: Participants that meet the initial qualifying criteria will visit the study 
center for a screening meeting. The participant and a member of the research team will 
meet in a private office to discuss the informed consent form. The informed consent form 
will be provided to the participant for their review, the study will be described in full detail 
and any questions the interested participant has will be encouraged and responded to. If 
they choose to participate in the study, they will be asked to sign the consent form 
providing written consent. The participant will be informed that even though they signed 
the consent form, their participation in the study is dependent on anthropometric 
measures and diet and medical questionnaire answers to determine if they meet the study 
criteria.  
 
If the participant provides consent, they will be provided with questionnaires including 
Automated Self-administered 24-hour Dietary Assessment Tool (ASA24®),and medical 
history. All collected samples  and data will be coded to maintain participant anonymity. 
We will give the participants a small volume of Study Product to screen for tolerance of 
the bitter tasting Study Products. We will also measure height, weight and body 
composition using a DXA scanner. Their enrollment for the intervention will be determined 
from the results. If the participant is eligible for the study and is still interested in 
participating then they will be randomized to a study product order and scheduled to return 
to the study center for the first testing visit. 

 
Testing Days: 
Participants will report to the study center in the morning of each Test Day. Compliance 
with pre-test instructions (fasted > 10h, no alcohol >24h, no exercise >24h, consumed 
pre-test food) will be confirmed by the Investigator. Participants will complete a baseline 
Beverage Tolerability Questionnaire (BTQ) and satiety visual analogue scale. 
Participants will be asked to completely void their bladder and a sample will be analyzed 
for hydration status. Participants with samples reading greater > 1.025 USG, and asked 
to don the Bluetooth heart rate monitor chest strap. A study team member will assist the 
participant with application of a continuous ketone meter into the back of the arm, this will 
be removed and replaced with a fresh sensor at Test Days at ~2-week intervals; the 
sensor will be removed 24 hours after the cessation of the last in lab testing bout. 
Participants will be given written instructions on how to remove and dispose of monitor. 
A trained member of the study team will insert an IV cannula into a vein in the antecubital 
fossa to allow for repeated blood sampling. The cannula will be flushed with a small 
volume of saline after each sample withdrawal to maintain patency. At the same time as 
all whole blood samples, we will also collect capillary blood samples from a finger for real-
time analysis of blood BHB and glucose concentration, using lancing device, 
commercially available test strips and a handheld monitor (KetoMojo, CA, USA). 
Participants will wear a fitted facemask connected to a metabolic cart for 10 minutes to 
collect measures of respiratory gas exchange. Participants will exhale once into a 
commercially available handheld breath acetone analyzer (Readout, MI, USA). Baseline 
blood sample, baseline respiratory gas measures and baseline breath acetone will be 
collected.  
 
Participants will then consume the Study Product that they were randomly allocated for 
that Test Day (details of Study Products below). Time of ingestion should be +/- 60 
minutes from the time established at Test Day 1; they will be given 5 minutes to consume 
the Product. After Study Product consumption, they will remain at the study center for 
~4h, with blood and breath sampling occurring at regular intervals (see Figure 2). A total 
of 7 whole blood samples (7x ~8 mL each= ~56mL) will be collected each Test Day. 
Capillary blood samples and breath acetone readings will be collected at the same time 
as whole blood samples. The fitted face mask will be worn for 10 minutes each hour to 
collect respiratory gas measures. Participants will be asked to collect all urine passed 
during the test in a provided container, and asked to fully void their bladder and collect 
the urine at the end of the test day. Participants will complete satiety questionnaires each 
hour, and will complete a BTQ at the end of the Test Day.   
 
Participants will be asked to minimize ambulatory movement during the Test Day. Non 
caloric beverages (i.e.,water) will be permitted ad libitum and intake volumes will be 
recorded. At the end of each Test Day, the heart rate monitor and IV cannula will be 

removed and a dressing will be applied to the cannula site. Participants will be given a 
snack to consume.   
 
Sample Processing and Analysis 
Blood Processing: Whole blood collected through the IV cannula will be either 1) 
immediately stabilized for AcAc analysis using a validated method, 2) processed to 
plasma (EDTA tubes), or 3) serum (clot activator serum collection tubes) and then snap 
frozen in liquid nitrogen for storage prior to analysis (details below). We will collect 
capillary blood samples for real-time analysis of blood BHB and glucose concentration, 
using commercially available test strips and a handheld monitor (KetoMojo, CA, USA), 
this will also allow comparison of ketone values obtained via different methods.  
 
Blood Sample Analytical Methods:  
• 
Ketones will be determined by multiple methods to provide a robust measure of 
ketosis and allow cross validation. Firstly: by capillary BHB concentrations 
obtained from a finger stick using a handheld device (KetoMojo, USA). Secondly: 
plasma ketones (AcAc, R-BHB and S-BHB) will be measured using ultra-
performance liquid chromatography – tandem mass spectrometry (LC-MS). 
Thirdly: Continuous Ketone Monitoring (see details below) 
• 
Plasma hormone (insulin, GIP, ghrelin) will be analyzed using commercially 
available ELISA assay kits (Cayman Chemical, USA).   
• 
Plasma metabolites (free fatty acids, alanine, lactate) will be determined using 
standard enzymatic assays, which have been extensively used at OSU.  
• 
Whole blood clinical chemistry will be performed using a commercially available, 
clinical grade handheld analyzer (iSTAT, Abott, USA) and cartridges which will 
deliver the following data: Sodium, Potassium, pH, PCO2, Urea Nitrogen, Glucose, 
Hematocrit, TCO2, HCO3, Base Excess, Anion Gap, Hemoglobin (EC8+, Abott, 
USA).    
 
Continuous Ketone Monitoring: We plan to incorporate the use of a novel continuous 
ketone monitoring (CKM) system developed by Abbott Diabetes Care. The ketone sensor 
in this CKM device is similar to the FreeStyle Libre continuous glucose monitoring (CGM) 
described previously25, but it has been modified using wired enzyme technology with BHB 
dehydrogenase chemistry. The sensor adheres to the back of the arm where it 
continuously samples interstitial fluid for quantification of BHB concentration. The sensor 
is worn for a period of 2-wk, three sensors will be used to cover all test days in this study 
(6 weeks). The first sensor will be inserted with assistance from the study team at the 
start of Testy Day 1, it will be checked every visit and replaced at Test Days following ~2-
week intervals. The sensor will be removed 24 hours after the cessation of the last in lab 
testing bout (Testing Day 9). Participants will be given written instructions on how to 

remove and dispose of monitor. Feasibility, stability and other quality control parameters 
of this CKM has been established26.   
 
Urine Participants will void their bladder on arrival and hydration status will be determined 
via urine specific gravity (USG) reporting <1.025 and all urine passed during the study 
will be collected; volume will be recorded. Water intake (permitted ad libitum) will be 
recorded. Urine ketone concentration will be measured using a modified version of the 
LC-MS method described above.  
 
Respiratory Gases: Respiratory gas exchange will be assessed using breath-by-breath 
gas exchange measurements of VO2 and VCO2 recorded every 30-sec.Participants will 
wear a fitted face mask connected to a metabolic cart for 10 minutes every hour  (TrueOne 
2400, ParvoMedics, UT, USA).  Participants will also complete one exhale into a high-
resolution portable breath acetone meter (Readout Health, USA) for determination of 
breath acetone. 
 
Heart rate and heart rate variability: At the start of each Test Day participants will don a 
Bluetooth chest strap that is sensitive enough to detect R-R intervals (Polar Electro, 
Finland). The chest strap will be worn for the duration of each Test Day and removed 
before the participant leaves the study center.  
Beverage Tolerability Questionnaire:  The BTQ used in this study is similar to that used 
in previous tolerability studies27,28,29,30 . Ten tolerability issues are included in the BTQ: 
gas/flatulence, nausea, vomiting, abdominal cramping, stomach rumbling, burping, reflux 
(heartburn), diarrhea, headache, and dizziness. Participants are asked if the issue was 
present (pre- beverage - baseline) or had occurred since they took the study beverage 
(post-beverage – 4h) at the following intensities: none, mild (awareness of symptoms but 
easily tolerated), moderate (discomfort enough to interfere with but not prevent daily 
activity) or severe (unable to perform usual activity). These correspond to scores of 0–3, 
respectively for each issue, giving a maximal composite score, defined as the sum of the 
ten items, of 30. 
Satiety Visual Analogue Scale: We will use a 3-item visual analogue scale, that assesses 
hunger, fullness and desire to eat by participant’s marking on a line anchored at either 
end with ‘not at all’ and ‘extremely.’ Distance along the line is measured in mm.   
STUDY PRODUCTS 
Background: The four exogenous ketone compounds (BHB Monoester, C6 Diester, AcAc 
Diester, (R) 1,3 Butanediol) will be used all have GRAS status as a dietary ingredient 
(Table 1). BHB Monoester, C6 Diester, and (R) 1,3 Butanediol are commercially available 
in the products ‘deltaG’ (TdeltaS Global, FL, USA), ‘Metabolic Switch’ (Juvenescence Ltd, 

NJ, USA) and ‘Avela’ (Genomatica, CA, USA), respectively. The AcAc Diester is 
commercially 
available 
(on 
Amazon 
https://www.amazon.com/KetoLogic-Ketone-
DiEster-Softgels) and is currently being used in clinical studies of Angelman’s 
Disease21.  We have been in communication with the companies that produce these 
exogenous ketones to ensure product availability and support for this work (see letters of 
support from BHB Therapeutics/Juvenescence and Disruptive Nutrition).  
 
Quality Control: As only small amounts of product are needed for this study, and products 
are shelf stable, our aim is that all product inventory will be acquired at the start of the 
study to ensure the same product lots are used throughout. We plan to obtain Certificates 
of Analysis to confirm product exogenous ketone content.    
 
Serving Size Rationale: Previous work has shown that lean body mass (LBM) is a major 
covariate in KE responsiveness9. Therefore, we will standardize exogenous ketone 
serving sizes to LBM (assessed using DXA) for all trials at 180 and 360 mg/kg LBM, which 
for a participant with 70 kg of lean mass corresponds to ~12.5 and 25 g, respectively. 
These serving sizes are representative of typical commercial serving sizes and are 
expected to elevate blood BHB in the range of 1.5 - 2 mM22,23. 
Blinding: It will not be possible to completely blind study participants or investigators due 
to visible differences between the C6 Ketone Di-ester and BHB-Mono-ester beverages 
and also the capsule delivery form for the AcAc Di-ester. However, study products will be 
coded (A, B, C, D and CON), and data analysts will remain blinded to the coding until 
analysis is complete.  
Storage: The study products will be stored in a dry secure location at ambient temperature 
(15- 77°F) and are best served chilled. Study product supplies are to be used only in 
accordance with this protocol and under the supervision of the Investigator.  
Table 1: Study product details 
Study 
Active 
Ingredient 
Serving Sizes 
Form 
Other product ingredients 
[ALLERGENS] 
Example 
Commercial 
Product 
(Company) 
BHB 
Mono-
ester 
180 
mg/kg 
and 
360 
mg/kg 
Ready to 
drink 
beverage 
Water 
DeltaG 
Tactical 
(TDeltaS 
Global, 
FL, 
USA)  

C6 Di-ester 180 
mg/kg 
and 
360 
mg/kg 
Ready to 
drink 
beverage 
Water, 
whey 
protein 
concentrate, modified gum 
acacia, 
natural 
and 
artificial 
flavors, 
cocoa 
powder, lecithin, fruit and 
vegetable juice for color, 
bisulfate of soda, caramel 
color, 
acesulfame 
K, 
cellulose 
gum, 
pectin 
(standardized 
with 
sucrose), 
sucralose, 
xanthan gum, canola oil 
[DAIRY AND SOY] 
Metabolic 
Switch 
(Juvenescence 
Ltd, NJ, USA) 
AcAc 
Di-
ester 
180 
mg/kg 
and 
360 
mg/kg 
1.5g 
capsules 
Geltain (bovine), glycerin, 
water.  
KetoLogic 
Ketone 
Di-
ester Softgels 
(Disruptive 
Nutrition LLC) 
(R)-1,3 
butanediol 
180 
mg/kg 
and 
360 
mg/kg 
Undiluted 
liquid 
ingredient 
– 
mixed 
on 
site  
with water 
and 
sweetener 
None  
Ketone-IQ 
(H.V.M.N. Inc, 
FL, USA) 
Control 
NA 
Beverage 
Water, canola oil, whey 
protein 
concentrate, 
modified 
gum 
acacia, 
natural 
and 
artificial 
flavors, 
cocoa 
powder, 
lecithin, fruit and vegetable 
juice for color, bisulfate of 
soda, 
caramel 
color, 
acesulfame K, cellulose 
gum, pectin (standardized 
with sucrose), sucralose, 
xanthan gum, canola oil 
[DAIRY AND SOY] 
National Food 
Laboratory, 
NY, USA 
 

DATA ANALYSIS AND STATISTICAL METHODS 
Primary outcomes  
• Difference in total plasma ketone appearance (AUC) between the two serving 
sizes of study products and control 
Secondary outcomes 
• Differences in other blood ketone kinetic parameters (peak concentration, time of 
peak concentration, AUC of different ketone bodies, capillary ketone 
concentrations). 
• Differences in urine and breath ketone excretion  
• Differences in blood metabolites (glucose, free fatty acids, alanine). 
• Differences in heart rate and heart rate variability and blood pressure 
• Differences in subjective tolerability and subjective satiety measured by 
questionnaires 
• Differences in blood hormones (insulin, ghrelin). 
• Differences in blood acid-base balance (bicarbonate, strong ions) 
• Differences in respiratory gas exchange (VO2 and VCO2) 
 
Statistical Plan. Statistical analysis of data will take place on completion of the study in 
collaboration with Buck Bioinformatics Core. Participant data will be included if they 
have completed at least 2 serving sizes of one exogenous ketone product AND the 
control Test Days (i.e., BHB- mono-ester 180 mg/kg, BHB mono-ester 360 mg/kg and 
Control). Any incomplete exogenous ketone compounds (i.e., participant completed C6 
Di-ester 180 m/kg and AcAc ester at 360 mg/kg) will be excluded from the analysis. All 
decisions regarding exclusion from the analysis population will be documented prior to 
database lock. Missing data will not be imputed, and only observed data will be included 
in the statistical analysis.   
 
Repeated measure group x time ANOVA (or a non-parametric alternative) will be 
performed following initial tests to ensure sphericity is not violated. We will compare 
data between control group vs. each individual exogenous ketone at  both doses (i.e., 
control vs BHB-mono ester (180 mg/kg) vs BHB-mono-ester (360 mg/kg). We will also 
test for condition x time interactions. If significance is detected, post-hoc comparisons 
will be made using Tukey’s HSD tests (or non-parametric alternative) to correct for 
multiple comparisons. Significance will be accepted at an alpha level of P < 0.05.  
 
Other data processing: 
• Area Under the Curve (AUC) will be calculated using the Trapezoid Rule. 
• Maximal/minimal concentration data (Cmax and Cmin) and time of 
maximal/minimal concentration (Tmax and Tmin) will be extracted from the 
raw data. 

• A composite tolerability score is calculated by adding the scores (0 -3, 
corresponding to none, mild, moderate, severe) of all ten listed symptoms. 
 
STUDY MONITOTRING 
Concomitant Medication/Supplements and Treatment 
All concomitant medications/supplements used 1 months prior to Screening Visit  and 
during the study will be reported to the study personnel for assessment and recorded in 
the participant’s study documents.  
 
Adverse Event Monitoring 
An AE is defined as any untoward medical occurrence in an investigation participant 
following written informed consent that does not necessarily have a causal relationship 
with the study product. An AE can be any unfavorable or unintended sign (including an 
abnormal finding), symptom, or disease temporally associated with the use of an 
investigational product, whether or not related to the investigational product. This 
includes any occurrence that is new in onset or aggravated in severity or frequency from 
the baseline condition, or abnormal results of diagnostic procedures (including 
laboratory test abnormalities).  
 
Some side effects or GI symptoms could occur as an outcome of these dietary 
interventions; side effects listed in the beverage tolerability questions and reported will 
not be categorized as AEs but recorded as study outcomes. Side effects, outside of 
what is expected as a result of study product consumption, reported by participants and 
judged by the Investigators as medically relevant events and related to study product 
will be recorded as AEs.   
 
Events should be considered AEs if they: 
• Result in discontinuation from the study, 
• Require treatment or any other therapeutic intervention, 
• Require further diagnostic evaluation (excluding a repetition of the same 
procedure to confirm the abnormality), 
• Are associated with clinical signs or symptoms judged by the Investigator to have 
a significant clinical impact. 
 
Grading and Severity 
The Investigator will evaluate all AEs with respect to their severity, and record the 
outcome and action taken on the AE study documents . AEs will be graded as: 
 
Mild:  
 Awareness of symptoms but easily tolerated  
Moderate:    Discomfort enough to interfere with but not prevent daily activity 
Severe:          Unable to perform usual activity 

 
Relationship 
The Investigator will also judge the likelihood that the AE was related to the study 
beverage and document this on the appropriate study documents as: 
 
NOT 
RELATED 
This category applies to those adverse experiences which, after 
careful consideration, are clearly and incontrovertibly due to 
extraneous causes (disease, environment, etc.). 
UNLIKELY 
 
In general, this category can be considered applicable to those 
experiences that after careful medical consideration at the time 
they are evaluated, are judged to be, unlikely related to the study 
beverage. 
POSSIBLY 
 
This category applies to those adverse experiences for which, 
after careful medical consideration at the time they are 
evaluated, a connection with the study beverage administration 
appears possible but cannot be ruled out with certainty. 
PROBABLY 
 
This category applies to those adverse experiences that, after 
careful medical consideration at the time they are evaluated, are 
felt with a high degree of certainty to be related to the study 
beverage.  
DEFINITELY 
 
This category applies to those adverse experiences which, the 
Investigator feels are incontrovertibly related to the study 
beverage.  
Appropriate therapeutic action and follow-up measures will be performed by the 
Investigator in accordance with appropriate medical practice standard of care.    
 
Serious Adverse Event Definition/Qualification 
A SAE is defined as an AE that results in any of the following outcomes: 
• Death (note that death is the outcome of a SAE and the cause of death should 
be listed as the AE), 
• Life-threatening event, 
• In-patient hospitalization or prolongation of existing hospitalization, 
• A persistent or significant disability/incapacity, 
• Congenital anomaly or birth defect, 
• Any other important medical event that may not result in death, be life-
threatening, or require hospitalization, may be considered a SAE when, based 
upon appropriate medical judgment, the event may jeopardize the participant and 
may require medical or surgical intervention to prevent one of the outcomes 
listed above.   
 

In the event of a SAE, the participant may be dropped from the study if the Investigator 
deems it necessary. 
 
Serious Adverse Event Reporting Instructions 
If in the opinion of the Investigator the event meets the criteria of a SAE the following 
procedures will be followed: 
• The Investigator will notify Institutional Review Board (IRB) of the SAE 
within the parameters and timeframe specified under the IRB Standard 
Operating Procedures (SOP). An initial report followed promptly by a 
complete report will be forwarded to the IRB, when applicable. 
• If a participant is hospitalized or hospitalization is prolonged due to the 
SAE, the hospital discharge summary will be obtained if possible. 
• If a death occurs and an autopsy is performed, a copy of the autopsy 
report will be obtained if possible.  
• All efforts must be undertaken to obtain follow-up information promptly. 
 
Recording of Adverse Events 
All AEs (AE or SAE) will be recorded on the AE study documents. For participants who 
have an ongoing AE at their final study visit, follow-up information will be captured in the 
AE eCRF page which will be completed after 30 days.   
 
Serious Adverse Event Follow-Up 
For all ongoing SAEs occurring during the study, the Investigator must submit follow-up 
reports regarding the participant’s subsequent course. All SAEs that are ongoing at the 
end of the study or upon discontinuation of the participant’s participation must be 
followed until either: 
• The event resolves, or 
• The event/condition has stabilized (e.g., in the case of persistent impairment), 
or 
• The event returns to baseline, if a baseline value is available, or 
• The participant dies, or 
• The event can be attributed to other than the study beverage, or to other than 
the study conduct. 
 
CONDUCT OF THE STUDY 
1. Ethics and Regulatory Considerations  
This study will be conducted according to Good Clinical Practice Guidelines, the 
Declaration of Helsinki (2004) and United State Code of Federal Regulation Title 21. 
Signed written informed consent for participation in the study will be obtained from all 
participants before protocol-specific procedures are carried out. Participants will be 

informed of their right to withdraw from the study at any time. Participants will be informed 
that their participation in the study is completely voluntary, personal information will be 
both deidentified to preserve anonymity.  
 
2. Institutional Review Board  
The Investigator will ensure that an appropriately constituted IRB, in compliance with the 
requirements of 21 CFR 56, reviews and approves the clinical study. Before the study is 
started, the Investigator will forward copies of the protocol and consent form for this study 
to the IRB for review and approval. IRB approval must refer to the study by exact protocol 
title and number, identify the documents reviewed, and state the date of review. The IRB 
must be informed of all subsequent protocol amendments. No alterations, modifications 
to IRB-approved documents, including the protocol, protocol summary, consent form, 
recruitment materials and questionnaires will be allowed. The IRB must also be informed 
of all SAEs and of unexpected AEs as outlined in the IRB’s SOPs or reporting guidelines.  
 
3. Informed Consent and Protected Health Information  
The study will be explained verbally as well as on the informed consent document. Each 
participant will be given ample opportunity to inquire about details of the study and to read 
and understand the consent form before signing it. It will be made clear that participants 
can withdraw from the study at any time.  
 
Each participant’s signed informed consent document must be kept on file by the 
Investigator. The participant should receive a copy of the informed consent document. A 
participant may not be admitted to the study unless informed consent of the participant 
(or his/her legally authorized representative) has been obtained.  
 
4. Participant Confidentiality  
The Investigator is responsible for ensuring that participants’ anonymity will be 
maintained. For all the data collected over the course of the study for each participant 
(i.e. records, biological samples and questionnaires) a unique subject identifier (i.e. a 
code) will be assigned and used instead of the subject’s name. The code for each 
participant which links the subject name with their identifier will only be available to 
research personnel. Electronic CRFs or other documents will identify participants by 
initials, number, or code, and not by name. The Investigator will keep a separate log 
showing codes, names, and addresses. Any records that contain the subject’s name and 
identifier will either be stored in the Kinesiology file storage room in a file cabinet (locked) 
or protected on a computer via password protection on the individual digital file and 
password protection on the computer the file(s) are stored on. All other records that 
contain the subject identifier only will also be kept in either a file cabinet in our locked file 
storage room or on a password protected computer. Subject names will never be used in 

any presentation or publication resulting from this study. The records will be maintained 
until the data are published and up to a maximum of ten years after the completion of the 
study. All records or biological data obtained after signing of the informed consent 
(including the screening visit, even for subjects that are not eligible for participation in the 
study) are treated with the same confidentiality safety measures as those subjects who 
qualify. Any information obtained during the prescreening for participants that were not 
eligible will be deleted 
 
5. Withdrawal of Participants from the Study  
Participants may be removed from the study for any of the following reasons:  
• 
A participant requests discontinuation;  
• 
The Investigator initiates removal for medical or compliance reasons;  
• 
Occurrence of any AE or condition that could, in the Investigator’s opinion, interfere 
with the evaluation of the effect of the study beverage or put the participant at 
undue risk.  
 
It is understood by all concerned that an excessive rate of withdrawals can render the 
study uninterpretable, therefore, unnecessary withdrawal of participants should be 
avoided. Should a participant decide to withdraw, all efforts will be made to complete and 
report observations as thoroughly as possible. In the event that a participant is withdrawn 
from the study, the reason for the withdrawal will be documented in the eCRF.  
 
 
 

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