Short Term Choline Supplementation and Cardiometabolic Health in Adults
Phase: NA
Conditions: Cardiovascular Risk Factor
Sponsor: Virginia Polytechnic Institute and State University

Cover page for Clinicaltrials.gov
Research Protocol
Official title: Short Term Choline Supplementation and Cardiovascular Health in Adults
NCT#: NCT04764162
Document date: 8/10/2021

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INSTRUCTIONS: 
●
Use this “TEMPLATE PROTOCOL (HRP-503)” to prepare a study protocol
outlining your research plan.
●
Depending on the nature of your study, some major sections might not be
applicable to your research. If so, simply mark as “N/A.” For example, a simple
survey might have many sections with “N/A.”  For subsections (e.g., 1.x or 8.x)
you can mark as “N/A”if you are certain that the subsection is not applicable.
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Download your current protocol from Protocol Management and indicate the
changes/revisions using the track changes feature in order to make review of the
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create a new paragraph wherever you need to make a change, and indicate
“Amendment: Date” before making a change to any section. Protocol
management will store the older versions of your protocol if the IRB or HRPP
staff need to compare them during the review.
PROTOCOL TITLE: 
Include the full protocol title.  
Short Term Choline Supplementation and Cardiovascular Health in Adults 
PROTOCOL NUMBER: 
Include the number assigned in Protocol Management (verify this has been added 
before submitting protocol to HRPP).  
18-535
PRINCIPAL INVESTIGATOR: 
Full Name and Degrees: Kevin P. Davy, PhD 
Department: Human Nutrition, Foods, and Exercise 
Telephone Number: 540-231-3487 
Email Address: kdavy@vt.edu 
FUNDING: 
Sponsor(s): National Institute of Health 
Funded already or in the proposal phase?: Awarded and new Award in progress 
Is Virginia Tech the primary awardee or the coordinating center of this grant or 
contract? If not, list the primary institution: Click here to provide a response. 

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VERSION NUMBER/DATE:  
Include the version number and date of this protocol. Versions should start at 1.0. 
 
1.0: 10/20/18 WIRB  
2.0: 1/30/2020 
3.0: 4/16/2020 
4.0: 8/10/2021 
 
REVISION HISTORY:  
Use this table to keep track of changes. Add more rows as needed. 
 
Revision 
# 
Version Date 
Brief Summary of Changes  
(i.e., the different sections) 
Consent 
Change? 
1.0  
10/20/18 
Initial submission to WIRB transfer to 
BRANY 
N 
2.0 
1/30/2020 
Change of Study Site Location to: 1872 Pratt 
Dr, Suite 1575 Garvin Bldg, Blacksburg, VA 
24060 
Y 
3.0 
4/16/2020 
New funding source, significant changes to 
protocol new measurements.  
Y 
4.0 
8/10/2021 
Changes to protocol, flyer and informed 
consent form 
Y 
 

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Table of Contents 
1.0 
Study Summary 
4 
2.0 
Objectives 
4 
3.0 
Background 
5 
4.0 
Study Endpoints 
6 
5.0 
Study Design and Statistical Analysis Plan 
7 
6.0 
Setting 
8 
7.0 
Study Intervention(s)/Investigational Agent(s) 
9 
8.0 
Procedures Involved 
11 
9.0 
Data and Specimen Long Term Storage and Use 
18 
10.0 Sharing of Results with Subjects 
20 
11.0 Study Timelines 
20 
12.0 Inclusion and Exclusion Criteria 
21 
13.0 Vulnerable Populations 
22 
14.0 Number of Subjects 
23 
15.0 Recruitment Methods 
24 
16.0 Withdrawal of Subjects 
25 
17.0 Risks to Subjects 
26 
18.0 Potential Benefits to Subjects 
30 
19.0 Data Management and Confidentiality 
30 
20.0 Provisions to Protect the Privacy Interests of Subjects 
32 
21.0 Provisions to Monitor the Data to Ensure the Safety of Subjects 
33 
22.0 Compensation for Research Related Injury 
34 
23.0 Economic Burden to Subjects 
35 
24.0 Consent Process 
35 
25.0 Process to Document Consent in Writing 
39 
26.0 Resources Available 
39 
27.0 Multi-Site Research 
41 
 
 
 

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1.0 
Study Summary 
 
Study Title 
Short Term Choline Supplementation and Cardiovascular 
Health in Adults 
Study Design 
2 x 4 crossover design with repeated measures and we will 
conduct descriptive univariate analyses on all study 
variables. 
Primary Objective 
To establish proof-of-concept for a greater reduction in 
glucose homeostasis following increases in gut microbiota-
generated TMAO on glucose homeostasis in older compared 
with young adults in order to conduct a larger, more 
comprehensive and mechanistic trial in the future. 
Secondary 
Objective(s) 
- Obtain preliminary data for effect size generation. 
Study Population 
We are recruiting men and women who are between the ages 
of 18-79 years of age. We will recruit individuals of all races 
and ethnic backgrounds. 
 
 
Sample Size 
60 subjects  
Research 
Intervention(s)/ 
Investigational 
Agent(s)  
-Interventions (Choline and Placebo supplementation in 
capsule form)  
Study Duration for 
Individual 
Participants 
How long will a participant spend in your study?  If multiple 
sessions, list time required for each session plus total.  
Visit: 1 = 1 hour  
Visit: 2, 8, 11, 17 = 3 hours each 
Visit: 3, 9, 12, 18 = 2 hours each 
Visit: 4, 10, 13, 19 = 3 hours each 
Visit: 5, 6, 7, 14, 15, 16 = 30 minutes each 
Total Time: 36 hours  
Acronyms and 
Definitions  
TMA = Trimethylamine 
TMAO = Trimethylamine-N-oxide 
T2D = Type 2 Diabetes 
GI = Gastrointestinal 
FMD = Flow mediated dilation 
CGM = Continuous glucose monitor 
 
 
 
2.0 
Objectives 
2.1 Describe the purpose, specific aims, or objectives of this study: 
 

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To establish proof-of-concept for a greater reduction in glucose homeostasis following 
increases in gut microbiota-generated TMAO on glucose homeostasis in older 
compared with young adults in order to conduct a larger, more comprehensive and 
mechanistic trial in the future. 
 
2.2 State the hypotheses to be tested:  
 
We hypothesize that gut microbiota-generated increases in plasma TMAO will be greater 
in older compared with young adults and this will be associated with a correspondingly 
greater reduction of insulin sensitivity, mixed meal tolerance, and 24-hr glycemic control 
in older adults. 
 
In addition, we hypothesize that the magnitude of change in gut microbiota-generated 
increases in TMAO and measures of insulin sensitivity, mixed meal tolerance, and 24-hr 
glycemic control with the intervention will be associated with the magnitude of change in 
the abundance of TMA-producing bacteria and/or expression of cutC TMA lyase. 
 
3.0 
Background 
3.1 Summarize the relevant prior research on this topic and gaps in current 
knowledge within the field of study: 
 
Dysbiosis of the gut microbiota, characterized as adverse changes in the abundance 
and/or composition of gut microbes, has been implicated in pathophysiology of a number 
of chronic diseases including neurological disorders, colitis, atherosclerosis, and 
metabolic diseases. Several lines of evidence in both animal models and humans also 
implicate gut dysbiosis in the etiology of insulin resistance and T2D. Future studies are 
needed to understand the potential link between gut microbial composition and function 
with glucose homeostasis and risk of T2D. Recently, gut microbiota-generated increases 
in TMAO has become a novel target for the treatment of cardiovascular diseases. The 
results of studies in rodent models indicate a causal role of TMAO in the development of 
atherosclerosis and several meta-analyses support a dose-dependency of TMAO in 
predicting adverse CVD events. Given the close relationship between T2D and 
cardiovascular disease, perhaps it is not surprising that a higher prevalence of T2D has 
been observed in populations with elevated TMAO. The potential role of TMAO in the 
development of insulin resistance and T2D is supported by multiple observations. 
Changes in gut microbial composition with aging are observed even in the absence of 
overt disease. However, the role of these changes on the reduced glucose homeostasis 
observed with aging is unknown. Interestingly, TMAO increases with advancing age in 
both mice and humans. Human TMAO increases are believed to be a consequence of 
changes in the gut microbiota, i.e., dysbiosis. Indeed, there is an increase in the 
abundance of TMA-producing bacteria with aging. Importantly, and as emphasized 
earlier, plasma TMAO concentrations are dependent on the composition of the gut 

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microbiota and higher baseline TMAO concentrations are associated with larger increases 
in TMAO with choline loading. As such, we hypothesize that older adults will 
demonstrate greater gut microbiota generated increases in TMAO following choline 
loading compared with young adults. Furthermore, the greater gut microbiota-generated 
increases in TMAO following choline loading will be associated with not only a greater 
abundance of TMA-producing bacteria but a larger decrement in insulin sensitivity, 
mixed meal glucose tolerance, and a reduction in 24-hr glycemic control. 
 
 
3.2 Describe any relevant preliminary data: 
 
N/A 
 
3.3 Based on the existing literature, provide the scientific or scholarly 
rationale for and significance of your research and how will it add to 
existing knowledge: 
 
The premise of the present proposal is that: 1) advancing age is associated with 
increases the abundance of TMA producing bacteria and TMAO concentrations; 2) older 
adults are at increased risk of insulin resistance, glucose intolerance, and T2D; and 3) 
there is accumulating evidence that elevated TMAO levels are predictive of, and 
contributory to, risk of T2D; and 4) the impact of gut microbiota-generated increases in 
TMAO on age-related changes in glucose homeostasis is unknown. Our proposal has 
significant translational potential as our findings will advance basic science findings in 
mouse models to determine relevance in humans and provide novel mechanistic insight 
into meta-organismal nutrient metabolism as a basis of age-related reductions in glucose 
homeostasis. One of the major challenges in understanding this link is the potential 
confounding influence of habitual dietary intake on key outcome variables. We will 
overcome this limitation by leveraging our existing expertise in rigorously standardizing 
participant diets to isolate the effects of meta-organismal metabolism of dietary choline 
as a basis for elevated risk of T2D with advancing age using state-of-the-art and 
reproducible methodology. Furthermore, our study will provide important insight into the 
gut microbiota as an important source of variability in glucose metabolism to TMAO. 
Our findings could have important policy and clinical implications. If modifying the gut 
microbiota to reduce age-related T2D risk is prudent, then clinical practice could be 
advanced by informing treatment guidelines and by providing rationale for 
individualizing nutritional or pharmaceutical interventions that target the gut microbiota 
as the interface between TMAO and T2D risk. 
 
 
4.0 
Study Endpoints 

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4.1 Describe the primary and secondary study endpoints. See links below for 
discussion of study endpoints and how they may differ from study 
objectives. These are most common in clinical trials but are sometimes 
applicable to other types of biomedical research, as well as social, 
behavioral, or educational research. See link below for a discussion. 
https://docs.google.com/document/d/1Wocz7K7a0hCQJPPO_khh5l1
SQQjhGDDGHzcOPRHR5Tw/edit?usp=sharing  
 
With this study, there are three continuous measurements which will define our study 
endpoints. These include oral glucose tolerance, flow mediated dilation of the brachial 
artery and aortic pulse wave velocity measurements corresponding to arterial stiffness 
and a mixed meal tolerance test. 
 
With regards to our hypothesis stated in section 2.2, our study endpoints will be as 
follows: Gut microbiota-generated increases in plasma TMAO will be greater in older 
compared with young adults and this will be associated with a correspondingly greater 
reduction of insulin sensitivity, mixed meal tolerance, and 24-hr glycemic control in older 
adults. 
 
 
4.2 Describe any primary or secondary safety endpoints. These should be 
included for all studies that are greater than minimal risk. (Minimal risk: 
The probability and magnitude of harm or discomfort anticipated in the 
research that are not greater in and of themselves than those ordinarily 
encountered in daily life or during the performance of routine physical or 
psychological examinations or tests.): 
 
Our safety endpoints will correspond to the potential risks that subjects may experience 
which are stated in section 17.1.  
Potential safety endpoints which could occur with this study are as 
follows: 1) Any severe GI distress or complaints reported with 
consumption of the choline or placebo supplement, 2) Discomfort or pain 
which is not tolerated by the subject with blood draws, 3) Discomfort and 
pain due to the inflation of the BP cuff on the forearm of the participant; 
and 4) Discomfort which is reported when measuring arterial stiffness 
measurements due to slight pressure being applied to the carotid, brachial, 
radial and femoral arteries. 
 
5.0 
Study Design and Statistical Analysis Plan 
5.1 Describe the basic study design/approach (e.g., qualitative study using five 
focus groups of first year students to describe assimilation into the 
university community; randomized controlled trial of a behavioral change 

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intervention to increase dietary intake of whole grains; pre- post-test 
evaluation of new pedagogical techniques to improve adult literacy): 
 
A double-blind randomized 2x3 crossover design with 3 repeated measures in each 
period will be used. Pre-/post-test evaluation used in each period to evaluate the ingestion 
of choline/placebo on the effects to cardiometabolic health. 
 
 
 
5.2 Describe corresponding data analysis plan/approach (e.g., content analysis 
of focus group transcripts; descriptive analysis followed by linear 
regression modeling; nonparametric analysis of pre- and post-test 
measures): 
 
Data will be examined for the presence of outliers, violations of normality and missing 
data. Major violations of normality will be corrected with an appropriate transformation 
procedure. In case of an outlier, rather than transform the data, the outlier will be 
“Winsorized”, that is, replaced by the most extreme value in the tail of the distribution.  
Data will be checked for sequence, period and carryover effects. Next, a mixed-effects 
linear model (using SAS PROCMIXED) will be used to estimate the treatment 
differences between choline and placebo (H0: μAB - μBA = 0). Because of the two 
repeated measurements of the dependent variable in each period, we will treat the 
multiple observations as nested within individuals. This will allow us to model the 
various sources of intra-person and inter-person variability, as well as account for the 
correlation in the nested data to make the correct inference regarding differences between 
treatments. Confidence intervals (95%) and bootstrapped standard error estimates will be 
reported for statistical precision and estimation of effect sizes for a larger more 
comprehensive trial. An identical approach will be used for analysis of other outcomes 
(e.g., arterial stiffness, etc.). 
 
6.0 
Setting 
6.1 Describe the sites or locations where your research team will conduct the 
research.  Consider each of the items listed below: 
● 
Identify where your research team will identify and recruit potential 
subjects. 
● 
Identify where the team will perform the research procedures. 
● 
Describe the composition and involvement of any community 
advisory board(s). 
● 
For research conducted in other locations, describe: 
o 
Site-specific regulations or customs affecting the research at 
those locations. 
o 
Local scientific and ethical review structure at those locations.  
Examples include work in other cultures or ethnic groups 
(within or outside of the U.S.) and work with churches. The 

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HRPP will provide additional guidance for international 
research. 
 
 
- Subjects will be recruited from the New River Valley and surrounding areas.  
- The study will be conducted in the Department of Human Nutrition, Foods, and 
Exercise at Virginia Tech, Blacksburg, VA. Participants will be required to come to 
Garvin Building, Suite 1575 (1872 Pratt Dr., Blacksburg, VA 24060) for 19 visits.  
 
 
 
7.0 
Study Intervention(s)/Investigational Agent(s) 
7.1 Describe the study interventions (including behavioral interventions) and/or 
investigational agents (e.g., drugs or devices) to be used in this study.  Consider 
each of the items listed below:  
● 
Drug/Device Handling: If the research involves drugs or devices, 
describe your plans to store, handle, and administer the drugs or 
devices so that they will be used only on subjects, and only by 
authorized investigators. 
● 
Describe whether any of the following will be used: microwaves, X-
rays, DEXA scans, general anesthesia, or sedation 
● 
If control of the drugs or devices used in this protocol will be 
accomplished by following an established, approved organizational 
SOP (e.g., Research Pharmacy SOP for the Control of 
Investigational Drugs, etc.), please reference the SOP in this section. 
 
 
This study will utilize the use of choline supplement (500mg BID capsules) and a same 
look/taste placebo supplement (500mg BID capsules) randomized in a double blinded 
trial as interventions. 
 
The choline and placebo supplements will be stored and locked away in the Human 
Integrative Physiology Lab and only members of the research team will have access to 
these capsules. Once subjects are required to be provided with the choline or placebo 
supplement, they will be provided to the participants in sealed containers which are 
assigned by subject ID. Only members of the research team will provide the supplements 
to the subject.  
 
The capsules will be produced in aseptic conditions in the Human Integrative Physiology 
Laboratory by members of the research team.  
 
DEXA scans will be used as part of a body composition analysis required on four visits 
(Visit 3, 9, 12, 18). The amount of radiation that a DEXA exam incurs is less than the 
amount permitted by the Food and Drug Administration (FDA) per year. The amount that 
will be received is equal to 4*1/20 of a chest x-ray.  

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7.2 List the name of all drugs (including any vitamins, supplements, herbs, or 
nicotine) to be used in the study.  Indicate whether they have FDA 
approval, and list any limitations for their use:  
 
Choline: Choline is a water-soluble vitamin-like essential nutrient that is available as an 
over-the-counter supplement. Adequate intake for adults is 550 mg/day for men and 
425 g/day for women. Tolerable upper intake level for adults is 3500 mg/day. Doses 
over the daily upper intake levels may cause side effects such as sweating, a fishy body 
odor, gastrointestinal distress, diarrhea, and vomiting. Some individuals might be 
allergic to choline or the other ingredients. Individuals who have known allergies to 
choline or other ingredients will be excluded. The subjects always will have the ability 
to terminate their participation for any reason without consequence. The dose of 1000 
mg/day dose of choline bitartrate USP (500 mg BID; NutriScience, Trumbull, CT) 
provided is well below the tolerable upper intake level for adults. Choline bitartrate 
USP is available as an over-the-counter supplement and is Generally Recognized as 
Safe by the FDA.   
Placebo: This will mimic the look of the choline capsule and contains 1000 mg (500 mg 
BID) of maltodextrin, a polysaccharide or carbohydrate commonly found in foods in a 
normal diet.  
Nitroglycerin: These are stabilized sublingual compressed tablets which will contain 
0.4mg of nitroglycerin and are FDA approved.    
 
7.3 List all devices, how they will be used, their purpose in the study, and if 
they will be used in a manner consistent with their approved uses. If they 
will be used in ways that are not yet FDA approved, indicate whether they 
need an IDE or a determination that they are exempt from the IDE 
Determination. If a determination of significant risk or non-significant risk 
is needed for any of the devices, include the researcher’s recommendation 
for each of those devices:  
 
- To perform the FMD portion of the investigation, duplex ultrasonography will used. To 
perform this, a GE Vivid S6 unit equipped with a high-resolution linear array transducer 
will be used in accordance with published guidelines. Furthermore, carotid imaging will 
be performed using the same unit to obtain carotid artery diameters. 
 

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- To perform the arterial stiffness measurements, applanation tonometry will be used. A 
fingertip probe which is equipped with a high-fidelity strain gauge transducer will be 
placed on the carotid, brachial, radial, and femoral arteries to obtain pressure waveforms 
and amplitudes. Again, this procedure will be performed following published guidelines.  
 
- Continuous measurements of glucose will be assessed using the FreeStyleLibre Pro, 
Abbott CGM. It will be placed on the upper back side of an arm. This will be performed 
following the guidelines and protocol from the device's company (Abbott).  
 
- Blood pressure measurements will be obtained using a mercury sphygmomanometer or 
automated blood pressure monitor and these measurements will strictly conform to 
American Heart Association guidelines. 
 
- Body composition analysis will be obtained via a DEXA scan with a Prodigy 
Advance, GE Healthcare unit. This will be performed only by trained and certified 
technicians and the amount of radiation that a DEXA exam incurs is less than the 
amount permitted by the FDA per year. 
 
7.4 If the drug is investigational (has an IND) or the device has an IDE or a 
claim of abbreviated IDE (non-significant risk device), include the 
following information: 
● 
Identify the holder of the IND/IDE/abbreviated IDE. 
● 
Explain procedures followed to comply with sponsor requirements 
for FDA regulated research for the following: 
 
Applicable to: 
FDA Regulation 
IND Studies 
IDE studies 
Abbreviated 
IDE studies 
21 CFR 11 
X 
X 
 
21 CFR 54 
X 
X 
 
21 CFR 210 
X 
 
 
21 CFR 211 
X 
 
 
21 CFR 312 
X 
 
 
21 CFR 812 
 
X 
X 
21 CFR 820 
 
X 
 
 
 
IDE exempt 
 
 
 
 
 
 
8.0 
Procedures Involved 

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8.1 Describe and explain the study design: 
 
Using a randomized cross-over design, participants will be given and asked to consume 
either choline or a placebo (maltodextrin) supplement for four weeks and will consume 
1000 mg (500 BID) of choline or placebo per day. Testing will take place over twelve 
visits. Participants will complete a screening visit and two periods of three-day testing 
sessions before and after the four-week period of choline and placebo supplement. In 
between experimental sessions, there will be a two-week “washout”.  
 
 
8.2 Provide a description of: 
● 
All research procedures being performed  
● 
If the study has more than one procedure, session, and/or subject 
population, describe each procedure, session, and/or study 
population separately. For complex studies, you are encouraged to 
include a figure or chart. 
 
 
Session 1: Baseline (screening) session (Approximately 2 hours) Garvin Building, Suite 
1575 
 
Overnight Fast:  Subjects will be asked to avoid eating or drinking anything except water 
and to avoid consuming any caffeine-containing beverages for 12 hours prior to this visit 
so that the test results will not be influenced by the food they eat or by the normal 
digestion process.   
 
Blood Chemistry: A blood draw will be collected for choline concentrations as well 
glucose, cholesterol, and other factors that may impact or be reflective of vascular 
function/health. A needle will be inserted in an arm vein to draw blood (approximately 2 
tablespoons/draw). 
 
Urine Test: Subjects will be asked to urinate in a small cup. Sodium and other 
electrolytes, glucose, protein, pH, and whether there are blood cells present will be 
measured to determine whether it is safe for each individual to participate in the study. 
 
Proof of COVID-19 vaccination: Subjects will be asked to bring their vaccination card 
against COVID-19 to the laboratory.  
 
Medical History:  Subjects will be asked to complete a medical history questionnaire, 
which will be used to screen for health problems (unstable heart disease) or other reasons 
(medications which influence study results) that would preclude participation. 
 

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Physical Activity: Subjects will be asked to complete a questionnaire regarding usual 
daily activities. They will also be asked to wear a device called an accelerometer over a 
4-day period to measure their physical activity level 
 
Infection/Inflammation Questionnaire:  Subjects will be asked to complete a 
questionnaire about any recent illnesses or infections that they may have had in the prior 
month. 
 
Dietary Record: Subjects will be asked to record/recall all of the food they eat during a 4-
day period. 
 
Body mass and height: Body weight will be measured on a digital scale accurate to +0.01 
kg. Height will be measured with a stadiometer 
 
Resting Blood Pressure and Heart Rate: Blood pressure measurements will be made 
under quiet, comfortable ambient laboratory conditions via mercury sphygmomanometry 
or an automatic blood pressure monitor. Heart rate will also be determined by an 
automatic blood pressure cuff. Measurements will conform strictly to American Heart 
Association guidelines. 
 
Session 2, 8, 11, 17: (Approximately 3 hours) Garvin Building, Suite 1575 
 
Overnight Fast: Subjects will be asked to avoid eating or drinking anything except water 
and to avoid consuming any caffeine-containing beverages for 12 hours prior to this visit 
so that the test results will not be influenced by the food they eat or by the normal 
digestion process.   
 
Resting Blood Pressure and Heart Rate:  Blood pressure measurements will be made 
under quiet, comfortable ambient laboratory conditions via mercury sphygmomanometry 
or an automatic blood pressure monitor. Heart rate will also be determined by an 
automatic blood pressure cuff. Measurements will conform strictly to American Heart 
Association guidelines. 
 
Body Mass: Body weight will be measured on a digital scale accurate to +/- 0.01 kg. 
 
Dietary intake analysis (24-hour recall): Subjects will be asked to recall all the food and 
beverages they consumed in the previous 24-hour period. 
 
Oral Glucose Tolerance Test:  One small plastic tube (catheter) will be placed in one of 
the subject's arm veins.  After a baseline blood draw to measure glucose, insulin, 
cholesterol, and other factors that may impact or be reflective of the subject's health 
status (e.g., C-reactive protein), the subject will drink a sugary drink of glucose (75 
grams) and blood will be collected in small amounts (less than one half teaspoon) every 
half hour (4 blood draws total) for a 2-hour period.      
 

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Stool Collection: Subjects will be given a test tube and asked to collect a stool sample. 
Subjects will be asked to bring the sample back to the laboratory during their next 
scheduled visit. All supplies and instructions will be provided by the laboratory. 
 
Continuous Glucose Monitoring: Glucose will be measured by a continuous glucose 
monitor (FreeStyle Libre Pro, Abbott). A small sensor will be placed on the subject's skin 
overlying the back side of their arm in order to measure glucose continuously. The sensor 
has a small plastic tube that is inserted into the tissue just below the skin and is held in 
place with sticky tape. The device will be worn for 3-4 days. 
 
Session 3, 9, 12, 18: (Approximately 2 hours) Garvin Building, Suite 1575 
 
Overnight Fast: Subjects will be asked to avoid eating or drinking anything except water 
and to avoid consuming any caffeine-containing beverages for 12 hours prior to this visit 
so that the test results will not be influenced by the food they eat or by the normal 
digestion process.   
 
Resting Blood Pressure and Heart Rate: Blood pressure measurements will be made 
under quiet, comfortable ambient laboratory conditions via mercury sphygmomanometry 
or an automatic blood pressure monitor. Heart rate will also be determined by an 
automatic blood pressure cuff. Measurements will conform strictly to American Heart 
Association guidelines. 
 
Body Mass: Body weight will be measured on a digital scale accurate to +/- 0.01 kg.  
 
Dietary intake analysis (24-hour recall): Subjects will be asked to recall all the food and 
beverages they consumed in the previous 24-hour period. 
  
Blood Chemistry: A blood draw will be collected for choline concentrations as well 
glucose, cholesterol, and other factors that may impact or be reflective of vascular 
function/health. A needle will be inserted in an arm vein to draw blood (approximately 2 
tablespoons/draw).  
 
Arterial Stiffness: Carotid Ultrasonography - Common carotid artery diameters will be 
measured from the image obtained from an ultrasound unit (GE Vivid S6) equipped with 
a high-resolution linear array transducer. Applanation Tonometry - The carotid, brachial, 
radial and femoral artery pressure waveform and amplitude will be obtained by a 
fingertip probe incorporating a high-fidelity strain gauge transducer. 
 
Brachial Artery Flow Mediated Dilation (FMD): FMD of the brachial artery will be 
assessed using duplex ultrasonography (GE Vivid S6) with a high-resolution linear array 
transducer according to published guidelines. Reactive hyperemia will be produced by 
inflation of a pediatric BP cuff around the forearm for 5 minutes. Offline analysis of 
baseline and post-reactive hyperemic diameters and velocities will be performed using 
edge detection software (Vascular Analysis Tools, Medical Imaging Applications, Inc.).  
 

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Endothelium Independent Vasodilation: This will be assessed by measuring brachial 
arterial dilation for 5 minutes following administration of 0.4 mg of sublingual 
nitroglycerine. Individuals will not be allowed to participate in this aspect if they take 
Cialis or Viagra or have a history of coronary heart disease. 
 
24-hour Urine Collection: Subjects will be asked to collect all urine samples over a 24-
hour period and bring the sample back to the laboratory during their next scheduled visit. 
All supplies and instructions will be provided by the laboratory. 
 
Body Mass and Composition:  Body weight will be measured on a digital scale accurate 
to +0.01 kg.  Height will be measured with a stadiometer. Percent body fat and fat-free 
mass will be measured in all subjects using dual-energy x-ray absorptiometry (DEXA) 
(Prodigy Advance, GE Healthcare).  
 
Pregnancy Test:  If a subject is female and pre- or peri-menopausal or not 
postmenopausal for at least 1 year, she will be required to provide a small cup of urine for 
a pregnancy test. 
 
Session 4, 10, 13, 19: (Approximately 3 hours) Garvin Building, Suite 1575 
 
Overnight Fast: Subjects will be asked to avoid eating or drinking anything except water 
and to avoid consuming any caffeine-containing beverages for 12 hours prior to this visit 
so that the test results will not be influenced by the food they eat or by the normal 
digestion process.   
 
Resting Blood Pressure and Heart Rate: Blood pressure measurements will be made 
under quiet, comfortable ambient laboratory conditions via mercury sphygmomanometry 
or an automatic blood pressure monitor. Heart rate will also be determined by an 
automatic blood pressure cuff. Measurements will conform strictly to American Heart 
Association guidelines. 
 
Body Mass: Body weight will be measured on a digital scale accurate to +/- 0.01 kg.  
 
Dietary intake analysis (24-hour recall): Subjects will be asked to recall all the food and 
beverages they consumed in the previous 24-hour period. 
 
Mixed Meal Tolerance Test:  Asmall plastic tube (catheter) will be placed in one of the 
subject's arm veins.  After a baseline blood draw to measure glucose, insulin, cholesterol, 
and other factors that may impact or be reflective of the subject's health status (e.g., C-
reactive protein), the subject will drink a mixed meal beverage (Ensure original plus ®, 
350 calories, 48g carbohydrate, 11g fat, and 16g protein) and blood will be collected in 
small amounts (less than one half teaspoon) at timepoints 0, 30, 60, 90, and 120 over a 
two-hour period.  
 
Continuous Glucose Monitoring: The device which was placed on the subject will be 
removed. 

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Choline Supplement/Placebo: Subjects will be given enough capsules to last one week 
and asked to consume 1000 mg (2 x 500 mg tablets) of choline or placebo with a bottle of 
water every day. 
 
Sessions 5-7, 14-16: (Approximately 30 minutes per session) Garvin Bldg, Suite 1575  
 
Choline Supplement/Placebo: Subjects will be given enough capsules to last one week 
and asked to consume 1000 mg (2 x 500 mg tablets) of choline or the placebo with a 
bottle of water every day. 
  
Resting Blood Pressure and Heart Rate: Blood pressure measurements will be made 
under quiet, comfortable ambient laboratory conditions via mercury sphygmomanometry 
or an automatic blood pressure monitor. Heart rate will also be determined by an 
automatic blood pressure cuff. Measurements will conform strictly to American Heart 
Association guidelines. 
 
Body Mass: Body weight will be measured on a digital scale accurate to +/- 0.01 kg.  
 
Dietary intake analysis (24-hour recall): Subjects will be asked to recall all the food and 
beverages they consumed in the previous 24-hour period. 
 
After session 10: Following this session, subjects will return to their normal activities for 
a two-week "washout". 
 
 
8.3 Describe: 
● 
Procedures or safeguards intended to reduce the probability and 
magnitude of risks. (For example: Reducing the risk of injury in a 
virtual reality study either by having the subjects sit during the study 
or by providing an obstacle-free space for walking.) 
● 
Be sure to describe all drugs and devices used in the research, when 
they will be administered or used, and their purpose.  
● 
Methods used to collect data about subjects. Please upload all data 
collection forms to Protocol Management. Some common examples 
are: 
● Screening questionnaires  
● Survey(s), including online surveys 
● Demographic questionnaire(s) 
● Interview guide(s), e.g., questions or pool of questions for semi-
structured interviews  
● Focus group guide(s) 
● Other documents used to collect data  
 

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- Blood Draw:  The P.I. or a trained technician will perform all blood draws. Aseptic 
conditions will be followed during all of the procedures. Universal precautions will be 
taken in collection and handling of all blood samples. Subjects will be informed that their 
blood will be analyzed for presence of HIV and Hepatitis, if an experimenter is exposed 
to their blood. 
 
- Endothelial Function: The risk of low blood pressure during nitroglycerine 
administration is minimized because the subject is already in the supine position.  There 
is a small risk of a headache, but this will last only a few minutes. Individuals taking 
Cialis or Viagra cannot participate in this aspect of the study. 
 
- DEXA scan: DEXA procedure will be performed by trained staff. Subjects will be 
informed of the risk of radiation exposure prior to study enrollment. 
 
- Continuous Glucose Monitor: The placement of the CGM on the subject will be 
performed by trained staff. This will take place in aseptic conditions, minimizing the 
risk of any infection. 
 
 
8.4 What data will you collect during the study and how you will obtain them? 
Please include descriptions of electronic data collection, database 
matching, and app-based data collection: 
 
- Study data will be collected on data sheets and manually entered into a database 
(Excel format) on a secure computer. 
 
8.5 Who will transcribe or code audio and/or video recordings?: 
 
N/A 
 
8.6 Include a description of any deception to be used in the study. Include 
justification for the use of deception (why the deception is necessary), 
describe the debriefing process, and describe how the study meets all the 
following criteria  for alteration of consent (deception is considered an 
alteration of informed consent): 
• The research involves no more than minimal risk to the subjects 
• The alteration will not adversely affect the rights and welfare of the 
subjects 

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• The research could not practicably be carried out without the 
alteration/deception 
• (Optional but encouraged in most cases) Subjects will be provided with 
additional pertinent information after participation (i.e., debriefing for 
studies involving deception)  
 
 
N/A 
 
 
8.7 If the study involves long-term follow-up (once all research related 
procedures are complete), describe what data will be collected during the 
follow up period and when it will occur:  
 
N/A 
 
 
 
 
 
9.0 
Data and Specimen Long Term Storage and Use 
9.1 If you will store data or specimens for future use, describe where you will 
store the data or specimens, how long they will be stored, and how and by 
whom the data or specimens will be accessed: 
 
All data will be stored in a locked cabinet in Dr. Kevin Davy's laboratory the Human 
Integrative Physiology Laboratory located in Garvin Building, Suite 1575 (1872 Pratt 
Dr., Blacksburg, VA 24060) which is also locked with access to authorized personnel 
only. The computer data will be stored in the locked lab on a computer that is password 
protected. All de-identified data will be kept indefinitely and there is no need to use 
specimens for the scope of the study. 
 
 
9.2 For specimens, list the data to be stored or associated with each 
specimen: 
 
N/A 
 

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9.3 Describe the procedures to release data or specimens outside of the 
research team, including the process to request a release, approvals 
required for release, who can obtain data or specimens, and what data 
will be provided with specimens: 
 
N/A 
 
9.4 Describe the identifiers to be included with stored data or specimens, as 
well as any key or code that could be used to make them identifiable. 
Describe where the code will be stored, who will have access to it, and 
when it will be destroyed: 
 
Codes using a combination of letters and/or numbers will be used to de-identify subjects 
from their personal information. This de-identified data collection sheet will be stored 
and locked away and the key will be kept in a locked cabinet separate from the subjects' 
completed data documents and accessibility will be limited to the PI and Co-PI's.  
The people who will have access to this data are the investigators and students involved 
in the study 
De-identified data may be kept indefinitely. Identifiable data and blood samples will be 
retained before being destroyed for 5 years. 
 
 
9.5 Please select the identifiers you will obtain (whether directly from 
participants or from another source), including but not limited to: 
 
☒ 
Name 
☒ 
Geographical subdivisions smaller than a state, including street address, 
city, county, precinct, zip code, and equivalent geocodes (note, the initial 
three digits of a zip code are not considered identifiable) 
☒ 
Elements of dates (except year) for dates directly related to an individual, 
including birth date, admission date, discharge date, date of death, and 
single year of age over 89 and all elements of dates (including year) 
indicative of such age (note, such ages and elements may be aggregated 
into a single category of age 90+) 
☒ 
Phone numbers 
☐ 
Fax numbers 
☒ 
Electronic mail addresses (e-mail) 
☐ 
Social Security numbers 
☐ 
Medical record numbers 
☐ 
Health plan beneficiary numbers 

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☐ 
Account numbers 
☐ 
Certificate/license numbers 
☐ 
Vehicle identifiers and serial numbers, including license plate numbers 
☐ 
Device identifiers and serial numbers 
☐ 
Web Universal Resource Locators (URLs) 
☐ 
Internet protocol (IP) address numbers 
☐ 
Biometric identifiers, including finger and voice prints (audio recording) 
☐ 
Full face photographic images and any comparable images (including 
video recording) 
☐ 
Student record number or identification number   
☐ 
User name for online or computer accounts 
☐ 
Any other unique identifying number, characteristic, or code (note this 
does not mean the unique code assigned by the investigator to code the 
data): Click here to explain.   
 
10.0 Sharing of Results with Subjects 
10.1 Describe whether you will share results (study results or individual subject 
results, such as results of investigational diagnostic tests, genetic tests, or 
incidental findings) with subjects or others (e.g., the subject’s primary care 
physician). If so, describe how you will share the results and include this 
information as part of the consent document. Upload materials you will use 
to explain the results to subjects: 
 
At the conclusion of the study or when the subjects' involvement in the study ends, they 
will be provided with their blood pressure readings, body composition results, and 
information on their dietary intake. This information will be provided in a summary of 
the study's findings.  
 
 
 
11.0 Study Timelines 
11.1 Describe: 
● 
The duration of an individual subject’s participation in the study (for 
example, 1 hour, 2-4 weeks, 3-5 years). 
● 
The amount of time expected to enroll all study subjects (weeks, 
months, years, etc.) 
● 
The amount of time expected for the investigators to complete this 
study including primary data analyses. 
 
 

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The duration of an individual's participation in this study will be approximately 12 
weeks, which will include 19 visits to the HNFE department at Virginia Tech and 
approximately 36 hours of their time. The actual time and frequency of the subject's visits 
will depend on their and the study staff's schedule. 
Subjects will begin the study on a rolling basis, but this will take place across 
approximately 12 months.  
The total amount of time expected to complete this study is approximately 24 months.  
 
 
12.0 Inclusion and Exclusion Criteria 
12.1 Describe how you will screen individuals for eligibility. When will 
screening occur and what procedures will you use? Upload any screening 
scripts or surveys to Protocol Management:  
 
 
Those who respond to the investigation's advertisements will be asked to complete a brief 
online screening questionnaire to confirm basic eligibility criteria on Qualtrics (e.g., age, 
health status).   
Furthermore, subjects will be screened for any possible health problems that would make 
it unsafe to participate, they will be required to complete a medical history questionnaire 
and made fully aware of their right to withdraw from the study at any time.  
 
 
 
 
12.2 Describe the eligibility criteria that define who will be included and who 
will be excluded from enrollment for each procedure of your study. 
Include any geographic criteria (e.g., Virginia Tech undergraduate 
students, a national sample of adults with engineering degrees, minors 
aged 8-12 in the New River Valley, university faculty in Virginia and 
Paris, France): 
 
Inclusion criteria is as follows: 
- 18-79 years of age 
- Sedentary (low physical activity) 
- Not diabetic 
- Not taking medications that influence the study results 
- Not have severe obesity (BMI <35kg/m^2)  
- Non-smokers 
- Not pregnant  
- Without any major medical problems  

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- Fully vaccinated against COVID-19. Individuals are fully vaccinated 2 weeks after their 
final dose of an approved vaccine. 
 
Exclusion criteria would be if individualss do not fall under any of the above criteria. 
Therefore, subjects will be excluded if they are not between 18-79 years of age, 
pregnant, a smoker, have a BMI >35 kg/m^2. Additionally, if they have changed their 
dietary patterns in the last month, have unstable heart disease or diabetes, vegetarians, 
vegans, untreated high cholesterol, or have health problems that make it unsafe to 
participate 
 
12.3 Indicate specifically whether you will include or exclude each of the 
following special populations: (You may not include members of these 
populations as subjects in your research unless you indicate them in the 
description of your subject population.) 
● 
Minors, as defined by state law where the study is performed 
(infants, children, teenagers) 
● 
Pregnant women (can be included in minimal risk studies by 
mentioning in section 13.1) 
● 
Prisoners (including all incarcerated individuals) 
● 
Adults not capable to consent on their own behalf 
 
All of the above will be excluded from the study. 
 
 
13.0 Vulnerable Populations 
13.1 If the research involves individuals who are vulnerable to coercion or 
undue influence, please describe additional safeguards you will include to 
protect their rights and welfare. Consider the applicable items listed 
below: 
● 
If the research involves Virginia Tech students, indicate whether 
these are students of any of the investigators. If so, describe whether 
the activities will take place during class time as part of the 
curriculum and the steps you will take to reduce the possibility that 
students feel obliged to participate in order to improve their course 
grade. The HRPP can provide further guidance as needed. Describe 
whether you will request access to student records (e.g., SAT, GPA, 
GRE scores). 
● 
If the research involves employees of Virginia Tech or the research 
sponsor, describe steps you will take to ensure that the employees 
are freely participating and describe how their data will be 
protected from inspection by their supervisors.  

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● 
If the research involves Virginia Tech NCAA athletes, you must 
obtain approval from the athletic department.  
● 
For research involving Montgomery County Public Schools, you 
must obtain county approval (after obtaining contingent Virginia 
Tech approval). Other locales have different requirements; please 
check on these and describe here. Approval is typically granted by 
the superintendent, principal, and classroom teacher (in that order). 
Approval by an individual teacher is insufficient. School approval, in 
the form of a letter or a memorandum should be uploaded as a 
supporting document. 
● 
If the research involves pregnant women, review “CHECKLIST: 
Pregnant Women (HRP-412)” to ensure that you have provided 
sufficient information in this protocol. 
● 
If the research involves prisoners, review “CHECKLIST: Prisoners 
(HRP-415)” to ensure that you have provided sufficient information 
in this protocol. 
● 
If the research involves persons who have not attained the legal age 
for consent to treatments or procedures involved in the research 
(minors), review the “CHECKLIST: Minors (HRP-416)” to ensure 
that you have provided sufficient information in this protocol. 
● 
If the research involves cognitively impaired adults, review 
“CHECKLIST: Cognitively Impaired Adults (HRP-417)” to ensure 
that you have provided sufficient information in this protocol. 
 
 
This research study has the potential to include employees of Virginia Tech. 
However, during the consenting process of the research study, the participants 
will be made aware that only members of the research study will have access to 
their data and that this data will utilize a coding system making their data 
unidentifiable. This data will be locked away and they will be made fully aware of 
their right to withdraw from the study at any time. 
 
 
 
14.0 Number of Subjects 
14.1 Indicate the total number of subjects to be enrolled and how this number 
was determined (e.g., sample size calculation [show], number of available 
subjects in a finite pool, number of tests funding award would allow):  
 
We have powered our pilot study to detect a change in insulin sensitivity 
of 20 ±32% (effect size=0.63).  We believe changes in other outcomes will 
be of similar magnitude.  We will enroll 60 participants.  Importantly, the 
purpose of this pilot study is to assess feasibility, acceptability, and 
provide proof of concept.  Even with a small sample size (n=60), a cross-

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over design with 4 repeated measures will give us fairly good estimates of 
the effect sizes, which is needed for a more comprehensive future trial. 
 
14.2 If this is a multi-site study, indicate the number of subjects to be enrolled 
at this site and the total to be enrolled from all sites: 
 
N/A 
 
14.3 If applicable, indicate the number of potential subjects you expect to 
screen for enrollment, and the number of subjects you will need to 
complete the research procedures: 
 
300-400 potential subjects will be screened for eligibility in this study. Of this 300-400 
individuals, 60 participants who are eligible will be enrolled to take part in the 
investigation.  
 
 
14.4 If the study has more than one procedure, indicate the total number of 
subjects to undergo each procedure separately: 
 
All subjects will complete each procedure. 
 
15.0 Recruitment Methods 
15.1 Describe when, where, and how you will recruit potential subjects: 
 
Recruitment will take place immediately following IRB approval. We are looking to 
recruit subjects from the New River Valley and surrounding areas. We will recruit 
subjects via posted fliers, newspaper advertisements, web postings, and community blood 
pressure screenings. Additionally, we plan to contact previous study participants/people 
who have expressed interest in future studies and who gave us permission to contact them 
for this purpose. 
 
15.2 Describe the source of subjects (for example, clinic patients with specific 
conditions, students in the library, community members at a gathering, or 
members of a local gym): 
 

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We want to recruit from the general population. 
 
15.3 Describe the methods that you will use to identify potential subjects: 
 
Posted fliers, newspaper advertisements, web postings, and community blood pressure 
screenings. Additionally, we plan to contact previous study participants/people who have 
expressed interest in previous studies who gave us permission to contact them for future 
studies. Potential subjects will also be filtered via the use of Qualtrics as a basic screening 
for eligibility and potential subjects to follow up on.  
 
15.4 Describe materials that you will be use to recruit subjects. Attach copies 
of these documents with this protocol in Protocol Management and be 
sure to include the IRB protocol number on each document.  
● For flyers, attach the final copy of printed flyers.  
● For Virginia Tech News, Facebook postings and ads, newspaper ads, 
websites, MTurk/SONA/online survey systems, etc., attach the final 
wording and graphics to be used.  
● For email recruitments, please include the subject line.  
● For advertisements meant for audio broadcast, please submit the 
wording of the advertisement prior to taping (to avoid having to re-
record with approved language) and submit the final recorded version 
for IRB review before use.  
● Describe any compensation to subjects. Separate compensation into 
appropriate categories, such as: reimbursement for expenses, time and 
effort, and additional incentives for study participation.  For each 
category, specify the amount (including any pro-rated amount), schedule, 
and method of payment. 
 
Subjects may receive up to $400 in compensation. 
 
Subjects will receive $20 for completing each testing session 2-18. After completing 
session 19, subjects will receive $60. There will be no compensation for completing the 
first screening session 
 
 
 
16.0 Withdrawal of Subjects 
16.1 Describe circumstances under which you anticipate subjects could be 
withdrawn from the research without their consent: 
 

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Circumstances which we anticipate that could result in withdrawal are if a subject fails to 
follow instructions or adhere to the investigations intervention, and if they are late or 
miss scheduled appointments.  
 
 
16.2 If applicable, describe any procedures for orderly termination (e.g., 
discontinuation of a study drug or debriefing after a behavioral 
intervention): 
 
If a participant is terminated or discontinued from the study for reasons as described 
above, the PI will mitigate issues leading to these problems. Additionally, the PI will 
discuss these difficulties with the participant and explain the importance of adhering to 
the intervention for the purpose of the study. The participant will be provided with the 
compensation that is due and provided their information which is available to them 
(blood pressure, body composition and dietary intake). It will then be suggested that the 
study personnel part ways with the participant.  
 
 
16.3 Describe procedures that you will follow when subjects withdraw from the 
research, including partial withdrawal from procedures with continued 
data collection (e.g., participant declines to continue with regular blood 
draws, but continues with periodic behavioral questionnaires): 
 
If a subject who is enrolled into the investigation is taking Cialis and/or Viagra, they will 
not be allowed to participate in the endothelial function aspect of the research which 
involves the sublingual use of nitroglycerin. However, they will be allowed to participate 
in all other aspects and the use of such medications will not be a determining factor in the 
inclusion/exclusion of the subject. 
 
In the rare chance in which there could be difficulty with the placement of the catheter in 
order to obtain blood draws for the oral glucose tolerance test, blood samples may then be 
performed via finger stick to collect glucose level measurements. 
 
If the subject is uncomfortable with any aspect of having blood drawn, they have the 
opportunity to voluntarily pull out of any/all of the blood draws and will be allowed to 
continue with other aspects of the study.  
 
 
17.0 Risks to Subjects 

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17.1 List the reasonably foreseeable risks, discomforts, hazards, or 
inconveniences to the subjects related the subjects’ participation in the 
research. Include for the IRB’s consideration a description of the 
probability, magnitude, duration, and reversibility of the risks. Consider 
physical, psychological, social, legal, privacy, and economic risks. Do not 
indicate “No risk” or “N/A.” Instead, for studies with very low risk (e.g., 
anonymous online questionnaire on a mundane topic) indicate “The 
investigators are not aware of any risks from participation in this study.” 
or “No more than risks than are found in everyday life.” The example 
consent form presents a tabular method for risk information, which you 
can also use here. Common risk types include: 
● Physical (e.g., potential for pain, discomfort, infection) 
● Psychological (e.g., potential for stress, discomfort, and/or 
embarrassment) 
● Social (e.g., potential for discrimination or stigmatization and 
disruption of personal and family relationships) 
● Legal (e.g., potential for disclosure of illegal activity, negligence) 
● Privacy (e.g., potential for personal information being accessed, used, 
or disclosed without the subjects’ knowledge or consent, breach of 
confidentiality/security) 
● Economic (e.g., potential for individuals to lose access to economic 
services, employment, insurability) 
 
Choline: Choline is a water-soluble vitamin-like essential nutrient that is available as an 
over the counter supplement. Adequate intake for adults is 550 mg/day for men and 425 
g/day for women. Tolerable upper intake level for adults is 3500 mg/day. Doses over the 
daily upper intake levels may cause side effects such as sweating, a fishy body odor, 
gastrointestinal distress, diarrhea, and vomiting. Some individuals might be allergic to 
choline or the other ingredients.  Individuals who have known allergies to choline or 
other ingredients will be excluded.  The subject always will have the ability to terminate 
their participation for any reason without consequence. The dose of 1000 mg provided is 
well below the tolerable upper intake level for adults.  
 
Blood Draw: Some pain or discomfort may be experienced when the catheter is inserted 
in the vein, but this should persist for only a short time.  During the blood draws, subjects 
may have pain and/or bruising at the place on their arm where the blood is taken.  In 
about 1 in 10 or 10% of the cases, a small amount of bleeding under the skin will cause 
bruising. The risk of a blood clot forming in the vein is about 1 in 200, while the risk of 
infection or significant blood loss is 1 in 1000.  There is a small risk of the vein becoming 
inflamed and/or painful in the hours or days after the needle is removed.  If subjects feel 
faint during or after a blood draw, they will be informed to notify the study doctor or 
study staff immediately and lie down right away to avoid falling.  Having staff who are 
experienced in venipuncture and blood draws will minimize these risks. 
 

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Oral Glucose Tolerance Tests: This procedure requires the placement of a catheter in an 
arm vein, the risks here are identical to that stated above. In addition, there is a small risk 
of low blood sugar occurring during or after the test. We will be monitoring subjects 
blood sugar frequently and can usually anticipate before blood sugar drops too low. If 
this happens, orange juice (with table sugar) or some other simple carbohydrate 
containing food will be provided. We will monitor the individual’s glucose until it returns 
to normal.  
 
DEXA Scan: The amount of radiation that subjects will receive in the DEXA exam is less 
than the amount permitted by the Food and Drug Administration (FDA) per year. The 
amount subjects will receive is equal to 1/20 of a chest x-ray. The more radiation an 
individual receives over the course of their lifetime, the more likely that individual’s risk 
increases in developing cancerous tumors. The radiation in this study is not expected to 
greatly increase these risks, however the exact increase in such risk is not known. 
 
HIV/AIDS:  In the event a researcher or other staff person is improperly exposed to the 
subject’s blood, his or her blood will be tested for the presence of HIV, the Hepatitis B 
Virus, and the Hepatitis C Virus. There will not be any cost to the subject for this test. 
The research team will follow proper procedures for testing and reporting as outlined by 
Virginia State Law, which includes sending the individual to a doctor's office for a blood 
draw. Should the blood be tested positive, the affected individual will be informed of the 
results and provided with the opportunity to receive appropriate and timely counseling. In 
addition, the results, if positive, will be sent to the local health department. 
 
Endothelial Function: Some pain or discomfort may be experienced when the blood 
pressure cuff is inflated and the participant may have pain and/or bruising at the place on 
the arm where the cuff was inflated but this is unlikely. However, these reactions are 
temporary and resolve within a short time after completing or stopping the procedure. 
There is a small risk that the subject will become lightheaded, dizzy, or faint following 
nitroglycerin administration. If these events occur, the feet of the subject will be elevated 
to alleviate the symptoms. Since they are already lying down, there is no risk of falling. 
These risks are minimized by having a trained individual perform the procedure.  
 
Arterial Stiffness: There is a risk of slight discomfort due to very slight pressure being 
applied to the carotid artery during the ultrasound procedure and to the carotid, 
brachial, radial, and femoral arteries during the tonometry procedure. 
Continuous Glucose Monitor: The placement procedure requires a sensor being inserted 
into the back side of the upper arm of the subject. When the wire-like sensor is deployed 
by the researcher, the subject can expect a sensation similar to a needle insertion for a 
blood draw. Potential risks associated here include discomfort during the insertion, pain, 
inflammation, redness, swelling, minor bleeding and minor infection at the site. These 
risks will be minimized by having a trained individual perform the procedure and this 
will be performed in aseptic conditions. Subjects might also experience these symptoms 
as a result of contact between the adhesive pad of the sensor and the skin. In rare cases, 
an infection can spread to other parts of the body. Allergic reactions can develop in 
response the adhesive used to keep the CGM in place. If these symptoms occur, subjects 

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have the ability to remove the CGM at-will and can anticipate that their symptoms will 
clear up within a short time. 
 
17.2  Indicate the measures you will use to minimize risks and monitor subjects 
for safety. (e.g., asking a subject at regular intervals to rate how they are 
feeling from 1 to 10, or to slowly crouch in order to check their balance.) 
 
Blood Draw:  The P.I. or a trained technician will perform all blood draws.  Aseptic 
conditions will be followed during all of the procedures.  Universal precautions will be 
taken in collection and handling of all blood samples.  Subjects will be informed that 
their blood will be analyzed for presence of HIV and Hepatitis if an experimenter is 
exposed to their blood. 
 
Endothelial Function: The risk of low blood pressure during nitroglycerine administration 
is minimized because the subject is already in the supine position.  There is a small risk 
of a headache, but this will last only a few minutes.  Individuals taking Cialis or Viagra 
cannot participate in this aspect of the study. 
 
DEXA scan: The DEXA procedure will be performed by trained staff. Subjects will be 
informed of the risk of radiation exposure prior to study enrollment. 
 
Whilst in the laboratory, each subject will be constantly monitored by the investigators 
and asked at regular intervals about how they are feeling. Furthermore, they will be 
made aware of their right to withdraw or discontinue any aspect of the protocol at any 
time. 
 
17.3 If applicable, indicate which procedures might have risks to the subjects 
that are currently unforeseeable. This will be rare, and usually applicable 
when testing a new drug or device or a new use of an existing drug or 
device: 
 
N/A 
 
17.4 If applicable, indicate which procedures might have risks to an embryo or 
fetus should the subject be or become pregnant: 
 
N/A 
 
17.5 If applicable, describe risks to others who are not subjects (e.g., collection 
of sensitive health data that might affect sexual partners if disclosed, 

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mandatory reporting of abuse, DNA testing that might affect family 
members or relationships): 
 
N/A 
 
18.0 Potential Benefits to Subjects 
18.1 Describe the potential benefits that individual subjects might experience 
from participating in the research. Include the probability, magnitude, and 
duration of the potential benefits, as this will be useful to the IRB’s 
risk:benefit analysis. Do not include benefits to society or others. Do not 
list monetary or non-monetary compensation for participation, as this is 
not a benefit These should be included in section 2 or 3 of this document: 
 
Subjects will receive information about their dietary intake, body 
composition and blood pressure. 
 
18.2 If applicable, specify that there are no anticipated direct benefits for 
participants:  
 
 
There are no direct benefits of participation. Subjects will be informed that this is not a 
wellness or medical exam and to discuss any concerns about their health information with 
their personal physician.  
 
19.0 Data Management and Confidentiality 
19.1 Describe procedures that you will use for quality control to ensure validity 
of collected data: 
 
To ensure quality control and validity of the data collected, all aspects of the research 
procedures will be conducted by trained technicians and all the procedures include 
reproducible techniques that are used on a regular basis by the PI's laboratory. 
 
19.2 Describe any existing data or biospecimens you will obtain as part of this 
study. Include:  
● 
Variables or samples to be obtained  
● 
Source of the data or specimens 

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● 
Your authorization to access or receive the data or biospecimens 
● 
Whether the data or biospecimens are publicly available 
● 
Whether the data or specimens you receive will contain identifiers  
 
N/A 
 
19.3 Describe the steps that you will take to handle and secure study data 
during data collection, storage, use, and transmission.  Include 
information about training of study staff, authorization of access, 
password protection, encryption, physical controls, certificates of 
confidentiality, separation of identifiers and data, etc.: 
 
Data will always be secured and deidentified. Only authorized and trained personnel 
will have access to study data. All data on electronic devices will be password protected 
in a locked office in a locked building. Study identifiers will be kept in a separate 
locked cabinet then data files.  
 
19.4 For multi-site studies, describe how data or specimens will be handled 
and secured for each site (e.g., central or disseminated data storage, data 
coordinating center): 
 
N/A 
 
19.5 Describe the plan for data disposition following the conclusion of the 
study (e.g., long term maintenance of data, data destruction methods).  
● 
What information will be included in the long term storage of data 
or specimens? 
● 
How long will the data or specimens be stored? 
● 
Where and how data or specimens will be stored? 
● 
Who will have access to the data or specimens during long term 
storage? 
● 
Who is responsible for receipt or transmission of the data or 
specimens? 
● 
How will data or specimens be shared or transported? 
● 
When and how will personal identifiers be destroyed? 
 
 
Only unidentified data or specimens will be stored, they may be stored indefinitely in the 
Garvin Building, Suite 1575. Only members of the research team in the protocol will 
have access and the PI is responsible for receipt and transmission of the data.  

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There is no intention to share or transport data at this current time. 
 
Personal identifiers will be destroyed after completion of all statistical analysis. 
 
 
 
20.0 Provisions to Protect the Privacy Interests of Subjects 
20.1 Describe the steps that you will take to protect subjects’ privacy interests. 
“Privacy interest” refers to a person’s desire to place limits on with whom 
they interact or to whom they provide personal information (e.g., 
collecting the minimal amount of private information required to complete 
the study, protecting the data once it is obtained):  
 
The minimal amount of private information will be collected and once the 
necessary data is collected all data will be unidentifiable through the use 
of codes and remain confidential in a locked cabinet. 
 
20.2 Describe steps that you will take to make subjects feel at ease with the 
research situation in terms of the questions being asked and the 
procedures being performed. “At ease” does not refer to physical 
discomfort, but the sense of intrusiveness a subject might experience in 
response to questions, examinations, and procedures (e.g., use of a same 
gender investigator to place sensors on the torso, a private changing area 
if clothing must be changed, sensitivity when discussing  pregnancy testing 
with subjects, making it clear on surveys that participants can discontinue 
at any time, not asking questions about private or sensitive issues unless 
necessary for the research): 
 
For procedures requiring the placement of sensors on a sensitive area of the body, such as 
tonometry measurements (arterial stiffness) on the inner thigh, this will be performed by 
a same gender investigator. 
 
There is a private changing area in our Human Integrative Physiology Laboratory for 
when individuals need to change into hospital gowns for the arterial stiffness and FMD 
measurements.  
 
There is a private bathroom for subjects to use and for when they are required to provide 
a urine sample for urinalysis. 
 
Sensitivity will be ensured when discussing pregnancy testing with subjects. 
During the consenting process and throughout the investigation, subjects will be made 
fully aware of their rights to withdraw from a procedure or the study. 

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20.3 Describe how you plan to access existing sources of information about the 
subjects (e.g., medical records, grades) and how you will protect 
participant privacy through the data security plan: 
 
No existing sources of information will be accessed. 
 
20.4 Describe any required reporting that might occur as a result of your 
research questions, study populations, and data collection methods. 
Examples for Virginia and Virginia Tech include: 
• Any suspicions (e.g., circumstantial, disclosed) of child abuse (physical, 
emotional, sexual) and neglect  
• Sexual discrimination and/or sexual violence that involves a student 
• Disclosure or signs of intention to harm oneself (i.e., suicidal ideation 
and/or plan) 
• Disclosure or signs of desire to harm others (i.e., homicidal ideation 
and/or plan) 
• Suspected abuse, neglect or exploitation of vulnerable adults (e.g., 
individuals with a disability, elderly persons) 
 
 
We do not anticipate any required reporting from the conduct of our study. However, we 
will report any of the above if there is circumstantial or disclosed information to the 
above points. 
 
 
 
21.0 Provisions to Monitor the Data to Ensure the Safety of Subjects 
Safety monitoring is required when research involves greater than minimal risk 
and is sometimes appropriate for other studies. 
21.1 Describe: 
● 
The plan to periodically evaluate the data collected regarding both 
harms and benefits to determine whether subjects remain safe  (e.g., 
periodic reporting to the IRB, establishing a data monitoring 
committee, reporting data monitoring committee findings to the IRB 
and the sponsor). 
● 
What data you will review, including safety data, unexpected events, 
and data that show the ability to produce the intended results. 
● 
How the safety information will be collected (e.g., with case report 
forms, at study visits, by telephone calls with subjects). 
● 
The frequency of data collection, including when safety data 
collection starts. 

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● 
Who will review the safety data and with what frequency. 
● 
The statistical tests for analyzing the safety data to determine 
whether harm is occurring. 
● 
Any conditions that will trigger an immediate suspension of the 
research (e.g., a serious adverse event). 
 
 
The data safety monitoring plan (DSMP) for this study focuses on close monitoring by 
the principal investigator (PI) in conjunction with a safety officer (Jose Rivero, MD), 
along with prompt reporting of excessive adverse events and any serious adverse events 
(AEs) to the NIH and to the Institutional Review Board. All serious AEs will be reported 
by the PI within 48 hours of occurrence to the IRB.  
  
Safety reports will be sent to the PI and to the safety officer.  The Project Coordinator 
(Elaina Marinik, Ph.D.) will be responsible for assembling the data and producing these 
reports as well as assuring that all parties obtain copies of these reports. Reports will be 
submitted annually to the VT IRB for review.  
  
The frequency of data review for this study differs according to the type of data, the 
availability of data collected, and the perceived level of risk. Subjects will be queried at 
the laboratory visits when supplements are provided as to any side effects or changes in 
health status that they experience. Any reported side effects will be discussed 
immediately with the PI and if necessary (i.e., serious adverse event), with Dr. Rivero. 
Out-of-range laboratory data (i.e., BP, bloodwork) will be discussed immediately with 
the PI and Dr. Rivero to determine if medical intervention or other actions are 
warranted. Subjects will be referred to their primary care physician if there are incidental 
findings. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
22.0 Compensation for Research Related Injury 
22.1 If the research involves more than minimal risk to subjects, describe the 
available compensation in the event of research-related injury, if any: 

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If a subject is injured as a result of this study, they will be informed to seek medical 
care. Neither the research team nor the University has money set aside to pay for any 
medical treatments that would be necessary if they are injured as a result of their 
participation in this study. Any expenses that they incur which includes emergencies 
and long-term expenses would be their own responsibility. They will be made aware 
that this limitation should be considered prior to beginning their participation in this 
study. 
 
22.2 Provide a copy of contract language, if any, relevant to compensation for 
research-related injury. At Virginia Tech, this is most common for 
sponsored research: 
 
N/A 
 
23.0 Economic Burden to Subjects 
23.1 Describe any costs that subjects might be responsible for because of 
participation in the research, including any uncompensated costs for items 
such as transportation, missed work, and childcare: 
 
There will be minimum transportation to and from the study location, but we do not 
anticipate any additional uncompensated costs. 
 
24.0 Consent Process 
24.1 Indicate the process by which you will obtain consent for study 
participation. Please upload all consent, parental permission, and assent 
forms, documents, and scripts referenced in this section to Protocol 
Management. 
Describe the following: 
● 
Where the consent process will take place (e.g., clinic waiting area, 
classroom, online) 
● 
The time interval between sharing the consent information with the 
prospective subject and obtaining consent. For lab, interview, and 
focus group studies, the Virginia Tech IRB prefers that subjects have 
at least 24 hours to review the consent form and study information 
before the appointment where consent will be obtained. For simple 
online survey studies, you can typically present the consent 
information immediately before subjects begin participation. 

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● 
If applicable, processes to ensure ongoing consent or assent (e.g., 
for multiple sessions; for research in which a minor will turn 18 
during the study; for longitudinal research with minors who will 
later be asked to provide or affirm their assent). 
● 
Please review “SOP: Informed Consent Process for Research (HRP-
090)” for recommended procedure. Describe your process, being 
sure to include: 
o 
The name and role of all study personnel who will be trained 
and certified by the PI to conduct the consent process 
o 
The time that will be devoted to the consent discussion 
o 
Steps that you will take to minimize the possibility of coercion 
or undue influence 
o 
Steps that you will take to gauge or ensure the subjects’ 
understanding 
 
Those who respond to the study's advertisements will be asked to complete a brief online 
screening to confirm basic eligibility criteria on uQaltrics (e.g., age, health status).  Those 
still interested and eligible to participate will be provided a consent document to review 
and consider.  Those who continue to express interest in participating will be invited to an 
initial session to hear the details of study and potential risks associated with their 
involvement.  They will be given a chance to ask any questions and have them answered 
to their satisfaction.  Those who would like to participate will be required to sign the 
consent document. 
A member of the investigative team will obtain consent from subjects.  Dr. Kevin Davy 
will be responsible for oversight of all aspects of the study 
The consent process will take place in the Human Integrative Physiology Laboratory in 
the Garvin Bldg and this will occur in the initial contact with the subjects, where the 
study will be explained to them and they will be emailed or receive a copy of the 
informed consent. 
Subjects will be allowed to take a copy of the informed consent home with them 
to review or a copy will be mailed, emailed or faxed to them.  They will return at 
a later date with the consent to ensure they have had enough time to review the 
consent and have any questions answered. 
 
Non-English Speaking Subjects 
● 
Indicate what language(s) other than English are understood by 
prospective subjects or representatives. 
● 
If non-English speakers will be recruited, describe the process you 
will use to ensure that the oral and/or written consent information 
provided will be in a language that they understand.  
● 
If you translate consent forms and study materials, please provide a 
certified translation of the form as well as the certification 
document. 

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● 
Indicate the spoken language that study personnel obtaining consent 
will use. Describe how you will assess fluency of personnel obtaining 
consent to ensure that the translation is accurate. 
 
 
Non-English-speaking individuals are not being recruited. English is the only language 
spoken by the study personnel (all native and fluent speaking). 
 
 
Waiver or Alteration of Consent Process (consent will not be obtained, 
required information will not be disclosed, or the research involves 
deception) 
● 
Review the “CHECKLIST: Waiver or Alteration of Consent Process 
(HRP-410)” to ensure you have provided sufficient information for 
the IRB to make these determinations (i.e., that it meets the criteria 
for a waiver or alteration of the consent process). 
 
N/A 
 
 
Subjects who are not yet adults (minors: infants, children, teenagers) 
● 
Describe the criteria that you will use to determine  legal age for 
consent to treatments or procedures involved in the research under 
the applicable law of the jurisdiction in which the research will be 
conducted (e.g., in Virginia, individuals under the age of 18 years). 
o 
For research conducted in Virginia, review “SOP: Legally 
Authorized Representatives, Minors, and Guardians (HRP-
013)” to determine which individuals in the state meet the 
definition of “minor.” 
o 
For research conducted outside of the state, please describe 
the legal requirements for the definition of “minor.”  
● 
Describe the process for obtaining parental permission.  
o 
Permission from one parent is acceptable for studies that 
involve no greater than minimal risk OR involve greater than 
minimal risk but present the prospect of direct benefit to the 
minor subject. 
o 
Permission from both parents is required in all other 
cases (unless one parent is deceased, unknown, 
incompetent, or not reasonably available, or when only 
one parent has legal responsibility for the care and 
custody of the minor). 
● 
Describe whether you will obtain permission from individuals other 
than parents or Legally Authorized Representatives, and if so, who 

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will be allowed to provide permission. Describe the process you will 
use to determine these individuals’ authority to consent to the 
minor’s general medical care. 
● 
Indicate whether you will obtain assent from all, some, or none of 
the minors. If you will obtain assent from some minors, indicate 
which minors will be required to assent. Consider chronological age 
and intellectual capacity when determining who will be required to 
provide assent (e.g., infants are unable to assent. However, 
teenagers are likely able to read and sign an assent form). 
● 
When assent of minors is obtained, describe whether and how you 
will document it.  Will minors sign an assent form or give verbal 
assent? 
● 
Attach parental permission and minor assent forms or scripts in 
Protocol Management.  
 
 
N/A 
 
 
Adults Unable to Consent  
● 
Describe the process you will use to determine whether an individual 
adult is capable of consent. 
● 
List the individuals from whom you will obtain permission in order 
of priority (e.g., durable power of attorney for health care, court 
appointed guardian for health care decisions, spouse, and non-
minor child). 
o 
For research conducted in the Virginia, review “SOP: Legally 
Authorized Representatives, Minors, and Guardians (HRP-
013)” to determine which individuals in the state meet the 
definition of “legally authorized representative.” 
o 
For research conducted outside of Virginia, please describe 
the legal requirements for obtaining permission from a legally 
authorized representative in the state where the research will 
occur. 
● 
Describe the process for assent of the subjects. 
o 
Indicate whether you will require assent from all, some, or 
none of the subjects. If some, indicate which subjects will be 
required to assent and which will not. 
o 
If you will not obtain assent from some or all subjects, please 
provide justification for not obtaining assent. 
o 
Describe whether and how you will document assent.  
 
N/A 

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25.0 Process to Document Consent in Writing 
25.1 Consult “SOP: Written Documentation of Consent (HRP-091)” for 
recommended procedures, and describe whether and how consent of the 
subject will be documented in writing: 
 
Subjects will be asked to sign the consent form to make sure they understand what is 
being asked from them and the potential risks and benefits involved. A member of the 
study team will acknowledge the witnessing of the signature with signing the consent 
also. 
 
 
 
25.2 If the research presents no more than minimal risk of harm to subjects and 
involves no procedures for which written documentation of consent is 
normally required outside of the research context, you can request that the 
IRB waive the requirement to obtain written documentation of consent 
(e.g., consent to participate is indicated by pressing a button for an online 
questionnaire – after the consent information is presented and before the 
questionnaire begins): 
 
N/A 
 
25.3 If you will document consent in writing, attach a consent document with 
places for signatures. If you will obtain consent, but not document consent 
in writing, please attach the consent script or text. Review “CHECKLIST: 
Waiver of Written Documentation of Consent (HRP-411)” to ensure that 
you have provided sufficient information. You should use “TEMPLATE 
CONSENT DOCUMENT (HRP-502)”to create the consent document or 
script: 
 
N/A 
 
 
 
26.0 Resources Available 
26.1 Describe the resources available to conduct the research. For example, as 
appropriate: 
● 
Describe the PI’s availability to supervise the research. 

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● 
Justify the feasibility of recruiting the required number of suitable 
subjects within the agreed recruitment period. For example, how 
many potential subjects do you have access to? What percentage of 
those potential subjects do you need to recruit? 
● 
Describe the time that you will devote to conducting and completing 
the research. 
● 
Describe your facilities. 
● 
Describe the availability of medical or psychological resources that 
subjects might need as a result of an anticipated or unanticipated 
consequence of participation in the research. 
● 
Describe your process to ensure that all persons assisting with the 
research are adequately informed about the protocol, the research 
procedures, and their duties and functions (e.g., training plans, 
detailed study notebooks). 
 
-  The PI commits 70% of his effort to research activities so is broadly available and in 
the laboratory on a daily basis.  
- We have powered our pilot study to detect a change in FMD  1.0±1.5%. The purpose of 
the pilot study is to assess feasibility, acceptability, and provide proof of concept. Even 
with a small sample size (n=60), a crossover design with  repeated measures will give us 
fairly good estimates of the effect sizes, which is needed for a more comprehensive future 
trial.  
- The PI has a 20+ track year record (15 years at Virginia Tech) recruiting and studying 
participants for studies similar to these. We typically need to screen 10 individuals for 
every 1 participant enrolled. 
- The PI oversees a laboratory with a clinical coordinator and 5-6 graduate students. He is 
in the laboratory on a daily-basis working with students and is involved in data 
collection.  
- The Human Integrative Physiology Laboratory (Director: K. Davy) is located in the 
Garvin Bldg affiliated the Virginia Tech campus in close proximity to the Laboratory for 
Eating Behaviors and Weight Management in Wallace Hall. There is ⁓1000sq ft of 
laboratory space dedicated to the conduct of the proposed studies. The clinical 
physiology laboratory (⁓250sq ft), the physiology laboratory (⁓250sq ft), body 
composition laboratory (⁓150sq ft), echocardiography laboratory (⁓150sq ft), and 
biochemistry laboratory (⁓250sq ft) will be used specifically for the conduct of the 
studies associated with the present proposal.   
- The laboratory staff are all BLS certified (including operation of an AED). The Virginia 
Tech Rescue squad is available (911) for on campus emergencies. Dr. Jose Rivero is the 
medical director of the laboratory and provides general oversight.  
- All staff are trained on study procedures they are assigned to perform before any subject 
is enrolled. The PI and clinical coordinator provide oversight and approve which staff 
have received adequate training. The laboratory staff meet weekly with the PI and clinical 
coordinator to discuss the ongoing protocol and research procedures and review each 
staff member's duties and responsibilities. Retraining/refresher training is performed 
periodically to avoid drift.   

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27.0 Multi-Site Research 
Contact the HRPP for multi-site research (involving multiple institutions) and 
the details required for this section will be provided. Otherwise, indicate N/A. 
 
N/A