Strategies to Augment Ketosis- Variations in Ketone Metabolism
Phase: NA
Conditions: Ketosis
Sponsor: Ohio State University

 
Strategies to Augment Ketosis 
Variations of Ketone Metabolism 
 
The Ohio State University IRB 
Approval June 5, 2023 
 
IRB# 2022H0292 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Protocol  
 
 
 
 
 

Strategies to Augment Ketosis: Variations in Metabolism (STAK-VKM) 
 
 
Title:  
Variations of Ketone Metabolism  
Short title: 
 
STAK-VKM 
 
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/External Collaborator: 
 
 
 
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  
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  
yr 
Year 
 
 
 

STUDY SYNOPSIS 
 
 
 
Study Title 
Variations of Ketone Metabolism  
Short Title 
STAK-VKM 
Study Design 
Open label, single-arm, observational study  testing blood 
ketone responses to C8 Ketone diester consumption across  
subjects of varying ages and metabolic statuses. 
Study Participants 
• Men and Women, ages 20-70 years old 
Planned Sample Size 
An evaluable sample of 336 participants/group is required; to 
allow for 20% attrition a final sample size of 400 is planned.  
Study Product 
1. C8 Ketone Diester 
 
Serving Sizes 
360 mg/kg of lean body mass 
Planned Study Period October 2022 – October 2025 
OUTCOMES 
Primary 
Difference in total plasma ketone appearance (AUC) across the 
metabolic ranges. 
Secondary 
• Differences in other blood ketone kinetic parameters (peak 
concentration, time of peak concentration, AUC R-BHB, 
capillary ketone concentrations).  
• Differences in ketone clearance rates via urine sample. 
• Differences in blood metabolites (glucose). 
• Differences in blood hormones (insulin). 
• Differences in tolerability and subjective satiety.  

SUMMARY AND BACKGROUND  
This project is focused on nutrition-based STRATEGIES TO AUGMENT KETOSIS 
(STAK), which enable people to increase blood ketone concentrations and to achieve 
‘nutritional ketosis’ which may have beneficial effects on resilience and health. Ketone 
esters (KEs) represent one promising STAK, these compounds consist 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). One KE 
compound is a diester of hexanoic acid (a ketogenic medium chain fatty acid) and (R)-
1,3 butanediol (C6 Di-ester). 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 use of KE products becomes more widespread, there is a critical need to expand 
our knowledge of how their metabolism varies between individuals to optimize dosing 
strategies for service members. Our goal is to determine the importance of age and 
metabolic healthy on blood ketone and glucose concentrations.  
 
KE Metabolism Varies Between Individuals.  This outcome of this study will help us to 
understand how the phenotype of the individual modulates the KE metabolic effect. 
Most studies of KE have been in homogenous populations, usually young, male 
athletes.  However, two striking experiments using identical, body weight adjusted KE 
doses in healthy and obese individuals found that BHB area under the curve (AUC) and 
removal was reduced by obesity and poor metabolic health. Similarly, ketone infusion 
experiments found that diabetes, obesity, and insulin resistance alter BHB metabolism. 
It is important to determine how obesity affects KE ‘sensitivity’ (i.e., breakdown and 
oxidation) because the increasing prevalence of obesity as a function of age. Age may 
be another important source of variation in ketone metabolism. The genes that control 
the ketone system are regulated by a cascade of transcription factors and hormones 
including PPARα and FGF21, which are themselves known to be affected by aging and 
dietary status, and the cellular protein sensor target of rapamycin (TOR). Aberrant 

hyperactivation of TOR with aging may reduce ketogenesis, while we observed that a 
long-term ketogenic diet 
specifically up-regulated 
PPARα activity. Our 
preliminary work 
revealed substantial 
changes across mouse 
lifespan in the 
expression of ketone-
related genes in the 
liver such as Hmgcs2 
(rate limiting for ketone 
production) and Bdh1 (rate limiting for BHB oxidation) between young, middle-aged, 
and old mice, with a nadir of gene expression in middle age before increasing again late 
in life (Fig 1A & 1B). We also found substantial age differences in response to matched 
doses of oral KE in mice (Fig 1C) and in rats. These data may have important 
implications for treating people of different ages and for translating KE technologies into 
the Department of VA. Therefore, we plan to study individual responses to KE ingestion 
across the lifespan, against the background of varying metabolic health 
 
 
IMPACT   
KEs could represent a practical STAK method that does not require any change in diet. 
However, there is a critical need to elucidate how KE 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 KE 
compounds based on individual characteristics. 
 
RESEARCH STRATEGY  
This is an open label, one arm study to characterize the effects of age and metabolic health 
on ketone responsiveness. We will enroll a large group representing a range of ages and 
metabolic health status.  N of 300 will be recruited and tested at the Physical Activity and 
Education services facility at the Ohio State University, Columbus, OH, and an N of 100 will 
be recruited and tested at the Buck Institute in Novato, California.  Data/sample collection 
will include blood (capillary and whole venous blood) and study product tolerability.   
 
OUTCOMES 
Primary  
 Figure 1 Change in mRNA expression for ketone oxidation enzyme (Bdh1) 
and production (Hmgcs2) between young, middle aged and old male mice. 
Blood BHB kinetics with a matched oral KE dose in young and old male mice.  

• 
Difference in total plasma ketone appearance (AUC) across various age and 
metabolic ranges 
Secondary 
• 
Differences in other blood ketone kinetic parameters (peak concentration, time of 
peak concentration, AUC of different ketone bodies, capillary ketone concentrations). 
• 
Differences in blood metabolites (glucose, free fatty acids). 
• 
Differences in subjective tolerability and subjective satiety measured by 
questionnaires 
• 
Differences in blood hormones (insulin).  
 
PARTICIPANTS 
This study aims to recruit equal numbers of male and female participants between the 
ages of 20 to 70yr. Thus, we will recruit a balanced sample of men and women with a 
range of ages (equal representation in each of the 5 included decades; 20-29yr, 30-
39yr, 40-49yr, 50-59yr, 60-70yr) and metabolic health via HbA1c clinical ranges 
(normal: <5.7, prediabetes: 5.7-6.4, diabetes: >6.4).  Each participant must meet all the 
inclusion criteria and none of the exclusion criteria at screening in order to participate. 
 
Inclusion criteria: 
• Ages 20 – 70 years 
• Participant is willing and able to comply with all study procedures including the 
following prior to Test Day: fasting (>10 h; water only), no alcohol (>24 h), no  
exercise (>24 h), no acute illness and controlled feeding before the 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 elucidate the effects of age and metabolic 
health status on plasma total ketone AUC. Using a prior human data from metabolically 
healthy and unhealthy groups, a 10% difference of BHB AUC (based on the 0.89 ± 0.26 
mM x hr values for young healthy people) corresponds to an effect size of 0.36 SD. Using 
this effect size with a type II error of 0.05, a sample size of 336 is necessary to detect the 
difference at 90% power. A total of 400 participants will be recruited to account for 20% 
drop off rate.  
 
Recruitment: 
Recruitment will occur through well-established methods at OSU, which include word of 
mouth, ResearchMatch database, social media engagement and flyers. 
  
 
METHODS  

Study overview: 
 
Figure 2 | Study schematic, showing timeline for a study day.   
 
Study Procedures 
Study Day 
Screening 
Test Day  
Visit to Study Center 
X 
X 
Informed consent 
X 
 
Medical intake questionnaire, diet assessment, 
anthropometrics1 
X 
 
HbA1c Measurement2 
X 
 
Submaximal Bike test3 
X 
 
Compliance assessment4 
 
X 
Hydration Status and Urine Collection5 
 
X 
Consume study product6 
 
X 
IV Cannulation and whole blood collection7 
 
X 
Tolerability assessment8 
 
X 
1. Medical intake questionnaire and diet assessment questionnaire are attached in Appendix . 
Anthropometric data will include: Hip and Waist circumference, BMI, height and weight. 
2. HbA1c measurement will be taken via fingerstick in order to stratify participants.  
3. A submaximal 6 minute bike test will be conducted to predict V02 Max (Astrand Test) 
4.  Prior to starting each test day, participants will be asked to confirm that they meet pre-test criteria, 
including: fasted >10h, no alcohol for 24h, no exercise for 24h and consumed the provided pre-test food.  
5. Prior to consumption of the Study Product, participants will be asked to completely void their bladder. 
And hydration status will be determined via urine specific gravity (USG) reporting <1.025. Participants 
will then void all urine into a provide container for the duration of the study visit.   
6. 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.   
7. 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 2. Cannula will be flushed with a 
small volume of saline after each sample to maintain patency 
8. Participants will complete a Beverage Tolerability Questionnaire prior to Study Product consumption 
and at the end of the Test Day (Appendix ) 
 
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 
HbA1C, Body Composition, diet assessment, 
submax bike test, study product palatability 

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 Product. We will also measure height, weight and body 
composition using bioelectrical impedance. Then we will use an A1CNow+, Hba1c Blood 
monitor kit to assess Hba1c score. We will use both body composition and Hba1c score 
to stratify participants to ensure various metabolic statuses are represented in the study 
population.  The participant will then complete a 6 minutes Astrand Bike Test to determine 
the predicted VO2 Max. This test does not require fasting and uses only submaximal effort 
upon the participant to determine cardiometabolic status by an average heart rate at 6 
minutes of pedaling at a moderate intensity. If the participant is eligible for the study and 
is still interested in participating then they will be scheduled to return to the study center 
for the testing visit. 
 
Testing Day: 
Participants will report to the study center in the morning of the 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). 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 will be asked to drink 160z of water and retest again in 30 
minutes.  Participants will be provided with a container to void all urine during the study 
visit. This will be aliquoted for urine R-BHB analysis to assess the clearance of ketones 
in the body. 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 then consume the C8 Ketone Diester Study Product. They will be given 
5 minutes to consume the Product. After C8 Ketone Diester consumption, they will remain 
at the study center for ~4-5h, with blood sampling occurring at regular intervals (see 

Figure 2). A total of 7 whole blood samples (~8 mL each) will be collected each Test Day. 
Capillary blood samples will be collected at the same time as whole blood samples. At 
the 4th hour finger stick, participants whose capillary ketones levels have not returned to 
baseline will be asked to stay for a 5th hour to assess the complete metabolism of the 
study product. 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 the Test Day, the 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 processed to 
plasma (EDTA tubes) or 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  and urine ketones (R-BHB) will be measured using ultra-performance 
liquid chromatography – tandem mass spectrometry (LC-MS).  
• 
Plasma hormone (insulin) will be analyzed using commercially available ELISA 
assay kits (Cayman Chemical, USA).   
• 
Capillary glucose concentrations obtained from a finger stick using a handheld 
device (KetoMojo, USA). 
Beverage Tolerability Questionnaire:  The BTQ used in this study is similar to that used 
in previous tolerability studies1516,17,18 . 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.   
Body Composition:   We will use the Quantum V Segmental BIA which provides 
quantitative regional body composition assessments that are similar to those produced 
by Dual-energy X-ray Absorptiometry (DXA) scanners. This is performed by using eight 
hand and foot electrodes (left and right side) with the subject in a supine position and 
measuring 13 resistance and reactance regions on the human body. 811 
 
STUDY PRODUCTS 
 
Background: The exogenous ketone compound, C8 Ketone  Diester, is commercially 
available in as the product ‘Metabolic Switch’ (Juvenescence Ltd, NJ, USA). 
 
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 360 mg/kg LBM, which for a 
participant with 70 kg of lean mass corresponds to ~ 25 g. These serving sizes are 
representative of typical commercial serving sizes and are expected to elevate blood BHB 
in the range of 1.5 - 2 mM 
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 Size 
Form 
Other product ingredients 
[ALLERGENS] 
Example 
Commercial 
Product 
(Company) 
C8 Ketone 
Di-ester 
360 mg/kg 
Ready to 
drink 
beverage 
Water, 
high 
fat 
whey 
protein 
concentrate, 
modified gum acacia, citric 
Metabolic 
Switch 

acid, soy lecithin, natural 
flavors, stevia leaf extract, 
pectin, 
sodium 
carboxymethyl cellulose, 
potassium 
sorbate. 
[DAIRY AND SOY] 
(Juvenescence 
Ltd, NJ, USA) 
 
DATA ANALYSIS AND STATISTICAL METHODS 
Primary outcomes  
• Difference in total plasma ketone appearance (AUC) between the ranges of age 
and metabolic status 
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 blood metabolites (glucose). 
• Differences in subjective tolerability and subjective satiety measured by 
questionnaires 
• Differences in blood hormones (insulin). 
 
Statistical Plan: Statistical analysis of data will take place on completion of the study in 
collaboration with Buck Bioinformatics Core. 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. To understand 
the relationship between BHB AUC and covariates such as age and metabolic health, we 
will first use multiple linear regression where BHB AUC is regressed on the covariates. 
P-values will be used to determine covariates that significantly predict BHB AUC. From 
the effect size of the coefficients, we can also assess which covariates have a greater 
impact on BHB AUC. The significance of interaction between covariates can also be 
investigated.  
To explore the pharmacokinetics of KE metabolism, we will develop a nonlinear mixed 
effect model (NLME). A one-compartment model with first-order absorption and 
elimination, where a constant proportion of ketone is absorbed/eliminated per unit time, 
has previously been observed to fit the pharmacokinetics of BHB Monoester 
metabolism19. The major goal of modeling the ketone data is to illustrate the relationships 
between the pharmacokinetic parameters such as Cmax, clearance(CL), volume of 
distribution(Vd) and the covariates. We will carry out preliminary analysis of the data after 
200 participants have completed the testing visit to determine effect size and determine 
if additional recruitment is required. Our anticipated results from this experiment are to 

show that blood ketone responses to a standard KE dose can be predicted based on age 
and metabolic health, and to inform individualized dosing guidelines.  
 
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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