Plasma and Urine Profiles of Blueberry Derived Polyphenols After a Short Term Intervention With Blueberry in Children With Typical Low Fruit and Vegetable Consumption.
Phase: NA
Conditions: Healthy
Sponsor: Arkansas Children's Hospital Research Institute

COVER PAGE 
Official title: Plasma and urine profiles of blueberry derived polyphenols after a short term 
intervention with blueberry in children with typical low fruit and vegetable consumption 
NCT04421950 
Last approved by the UAMS IRB: 09/03/2021 
 

Title: The Blueberry Study 
PI: Eva C. Diaz M.D.  
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Version 15 
9/3/2021 
 
IRB # 260493 
 
Study Title: 
The Blueberry Study 
 
 
 
Principal Investigators:  
Eva Carolina Diaz Fuentes M.D. 
15 Children’s Way, Slot # 317 
Little Rock, AR 72202 
Telephone: 501-364-3056 
Email: EDiazfuentes@uams.edu 
 
Co-investigator: 
Elisabet Borsheim Ph.D. 
15 Children’s Way, Slot # 317  
Little Rock, AR 72202 
Telephone: 501-364-3053 
Email: EBorsheim@uams.edu 
 
 
 
  
 
Study location: 
Arkansas Children’s Nutrition Center 
 
 

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Background and Rationale  
Polyphenols are bioactive compounds that have gained research interest due to their 
protective effects against non-communicable diseases (NCD) (1,2). Blueberries are a rich 
source of polyphenols, particularly, anthocyanins (ACNs) (3).  Their consumption has been 
linked with improved glucose levels in individuals at risk of type 2 diabetes (4), decreased 
blood pressure and arterial stiffness in women with hypertension, and decreased pain and 
disability in children with autoimmune arthritis (5). In addition, animal and in vitro studies 
from the Arkansas Children’s Nutrition Center (ACNC) have shown that 2 weeks of wild 
blueberry consumption improves bone health by preventing loss of bone collagen  by 
inhibiting bone forming osteoblast cell senescence (6).   
However, the mechanism by which blueberry derived polyphenols exert their 
protective effects  cannot be explained by their bioavailability as their systemic absorption  
is poor (7).  In the past decade, studies have shown that dietary polyphenols undergo 
extensive metabolism by the gut microbiota and the liver, giving origin to bioactive 
derivatives that potentially mediate health benefits (8).  To our knowledge, no study has 
characterized polyphenol derivative profiles in blood and urine in children after wild 
blueberry supplementation.  
LFV is now considered among the top 10 risk factors for the development of NCDs 
and overall mortality (9). Alarmingly, in the USA only 40% and 7% of children meet the U.S. 
Department of Agriculture Food Patterns fruit and vegetable intake recommendation, 
respectively (10). It is not known if a polyphenol rich intervention with wild blueberry can 
improve markers of immune, cardiovascular and bone health in children with low fruit and 
vegetable consumption (LFV). Data from ACNC suggest that fitness (peak VO2) modifies the 
association between fruit and vegetable (FV) consumption and vascular health of children. 
Specifically, children with combined low FV intake and low peak VO2 have higher blood 
pressure values than children with low fitness but higher FV consumption. These results 
suggest, that children with low fitness would not only benefit from more exercise but also 
from a higher FV content in their diets.  However, higher quality evidence is needed to inform 
clinical practice and dietary guidelines. 
 

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Objectives 
1) Characterize plasma and urine profile of polyphenol-derived metabolites of school-
age children supplemented short-term with wild blueberry powder. 
2)  Assess the effects of short-term wild blueberry supplementation on immune 
responses as well as on markers of bone metabolism and cardiovascular health in 
apparently healthy children who consume ≤2 cups of fruits and vegetables per day.  
3) Evaluate if cardiorespiratory fitness (peak VO2) modifies the association between 
blueberry derived polyphenol consumption and markers of cardiovascular health. 
 
Study Design and Procedures  
This is a double-blind randomized control trial 
aiming to characterize the profile of polyphenol-
derived metabolites in plasma and urine of 
children ages 11 to 12 years supplemented 
short-term with wild blueberry powder. Further, 
the 
study 
will 
assess 
if 
a 
short-term 
supplementation (up to 6 days) with wild 
blueberry powder impacts immune responses as 
well as markers of cardiovascular and bone 
health while assessing for the effect modification 
of peak VO2. Up to 44 children (boys and girls) 
will be recruited to participate. After telephone 
screening, the children and parents will come to 
ACNC for assenting/consenting and initial measurements. After this, children and parents 
will attend to 2 more study visits at ACNC (total of 3 study visits). A description of study 
procedures follow below. Children will be asked to stop taking any kind of nutritional 
supplement (e.g., multivitamins, vitamin D, fish oil, probiotics, prebiotics, immune boosters, 
and others) at least 2 weeks prior to the 1st study visit. Children who had an infection that 
required antibiotics will be scheduled after 2 months of finalizing antibiotic treatment. 
N=44 children (11 to 12 years old)
LFV consumers
n=22 placebo and n=22 wild blueberry
1st visit: assent/consent, dietary intake, 
anthropometrics, vital signs, DXA, peak 
VO2, low polyphenol diet education.
2nd visit:  Baseline blood, stool, urine, saliva 
collection. Vital signs and flow mediated dilation 
(FMD). low polyphenol diet. Beginning of 6-day 
intervention (wild-blueberry vs. placebo). 
3rd visit: blood, stool, urine, and  saliva 
sampling. Anthropometrics, vital signs, and 
FMD. blueberry/placebo containing 
product,
Figure 1: Study design

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Children with a viral infection such as diarrhea, cold or flu will be scheduled after 2 weeks of 
resolution of symptoms. 
Study visit (s) and assessments:  Three (3) study visits will be conducted at ACNC after an 
overnight fast.  All assessments may be repeated if/when needed, and if the participant is 
willing.  Following phone screening, qualifying participants will attend ACNC for further 
assessment of eligibility, informed consent and assent. Visit #1 will last up to 4 hours, visit 
#2 up to 4 hours, and visit #3 up to 8 hours. Visit #2 will take place if the participant has 
adhered to a low-polyphenol diet 2 days prior to the study visit. Adherence will be 
determined by research staff using the dietary logs provided to participants during visit #1. 
If participants have not been compliant, they will be given the option to re-schedule visit #2 
upon completion of 2 days on a low-polyphenol content diet.  The ACNC is a Well-Child 
facility separated from the ACH facilities with a separate entrance and is not used for clinical 
care which will limit exposures of the participants to illnesses.  Parents will be instructed not 
to come to the study visit if they or their child is ill.  The study visit will be rescheduled if that 
is the case to a time when both parent and child are well and asymptomatic.   
Study measurements and procedures 
Dietary Intervention 
 Low 
polyphenol 
Study 
Diet: Participants will be 
asked to adhere to a low 
polyphenol diet (i.e., less 
than ≤0.5cups of fruit and 
≤0.5 cups of vegetables per 
day, and avoiding cocoa 
products, 
nuts, 
sweet 
potato, 
whole 
grains, 
caffeinated drinks, tea, and 
others).  All participants will be instructed to follow a low polyphenol diet (a sample meal 
plan will be provided).  Participants will start the low polyphenol diet 2 days prior to visit 
#2 up until the day prior to visit #3 (total of 7 consecutive days on a low polyphenol diet).   
Table 1: Summary of study procedures 
Procedure/measurement 
Visit 1 
Visit 2 
Visit 3 
Low polyphenol diet 
 
x 
x 
Wild blueberry/placebo 
 
x 
x 
Medical History 
x 
 
 
Anthropometrics 
x 
 
x 
DXA 
x 
 
 
Vital Signs 
x 
x 
x 
Peak VO2 
x 
 
 
Dietary Intake 
x 
x 
x 
Study diet coaching 
x 
x 
 
Stool, 
urine, 
and 
saliva 
collection 
 
x 
x 
Endothelial function 
 
x 
x 
Blood draw 
 
x 
x 

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Participants will still be eligible if they consume less than or equal to two (2) cups of fruits 
and vegetables combined per day before Visit #2.  Visit 3 will continue even if the participant 
did not adhere to the low polyphenol diet.   
Wild blueberry/placebo intervention: wild blueberry containing foods (i.e., wild blueberry 
powder or wild blueberry extract) or identical placebo-containing foods will be consumed 
starting on the day of study visit #2 until the morning of visit #3 (total of 6 days on blueberry 
or placebo).  These food items have been designed in collaboration with the University of 
Arkansas Department of Food Science and may consist of oatmeal bars, beverages, ice pops, 
and cookies. Participants will receive product combinations that deliver up to 30g of wild 
blueberry powder per day (or the equivalent anthocyanin content in the case of products 
containing wild blueberry extract) or placebo. Parents and participants will keep a daily log 
which will be used to assess compliance with blueberry/placebo containing products and 
low polyphenol diet. Research staff may contact study participants between visits via phone, 
email, or text to inquire about their experience, perceived barriers with adhering to the 
intervention and to their goals. Participants will be asked to complete the Study Product Log 
book of what was consumed.  
Study Diet Coaching: Participants will be educated by a trained nutritionist on how to avoid 
polyphenol rich foods. A sample low polyphenol meal plan will be provided to the 
participants if needed.  
Medical History: an interviewer-administered medical history questionnaire will be 
conducted. 
Anthropometrics: Anthropometric measurements (body weight, height, waist and hip 
circumference) will be obtained using standardized techniques, while the children are 
wearing light clothing and without shoes. All anthropometric measures will be made in 
duplicate, or triplicate if not within acceptable agreement. 
Body Composition:  Body composition will be assessed using Dual-Energy X-ray 
Absorptiometry (DXA; Horizon-A with Advanced Body Composition™, Hologic, Bedford, 
MA).  Measures of body composition are needed to standardize peak VO2 (see below) to lean 
body mass content. DXA technology involves very small amounts of ionizing radiation. Total 
body fat and fat free masses, bone mineral density, bone mineral content (whole body, femur, 

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and spine) will be determined. This will use the newest DXA technology (Analysis with 
InnerCore™ Visceral Fat Assessment) which can separate the adipose tissue depot into 
visceral and subcutaneous fat mass by performing a scan of <3 min. 
Vital signs: Blood pressure, heart rate, and body temperature will be assessed using 
standard techniques at each visit. 
Fitness test (peak VO2): A graded exercise test will be performed to determine peak oxygen 
uptake (peak VO2). A bicycle fitness test will be conducted. During this test, the resistance of 
the stationary bicycle is gradually increased. The children will be told that they are free to 
stop the test at any time if they do not want to continue, but will be encouraged to perform 
maximally. During the test, the children will be required to wear an indirect calorimetry face 
mask in order to assess their oxygen uptake (VO2), carbon dioxide production (VCO2), and 
respiratory exchange ratio (RER; VCO2/VO2). Heart rate monitors will be worn to measure 
heart rate during the exercise test. Ample time will be given for familiarization with the 
stationary bicycle, face mask, and heart rate monitor. Children will be educated in how to 
assess their exertion levels, and will be asked to provide a rating of self-perceived exertion 
every minute during the test using the children’s OMNI scale. This is a numerical scale from 
0 to 10, with a score of 2 indicating “a little tired” and a score of 9 indicating “very, very tired,” 
with associated pictures to represent perceived physical effort. Peak VO2 will be determined 
by the combined assessment of: heart rate of ≥185beats per min, and/or respiratory 
exchange ratio ≥ 1.0; and/or ratings of perceived exertion on the children’s OMNI scale ≥ 8. 
The bike test will be conducted primarily by an ACSM registered clinical exercise 
physiologist (with M.Sc. in exercise physiology) with expertise in these procedures. Other 
research staff that have been trained in these measurements and certified by Dr. Elisabet 
Borsheim (Director of the Physical Activity Core at ACNC) may also conduct these 
measurements.  
 

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Dietary intake:  A 
food 
frequency 
questionnaire 
will 
be completed at visit 
1. 
Parents 
(or 
caregivers) will be 
asked to keep a log 
of what their child 
eats while on a low 
polyphenol diet (7 
days). Instructions will be provided to parents verbally, and/or via mail, and/or e-mail. Logs 
will be reviewed to assess compliance with the study diet before Visits 2 and 3.   
Stool, urine, and saliva sample collection: Stool samples will be collected and may be used 
to analyze the microbiome, polyphenol content, and polyphenol derived metabolites (table 
1). Parents will be given the option to collect the child’s stool sample at home. Instructions 
and supplies will be provided. Urine will be collected to measure wild-blueberry derived 
metabolites, markers of bone metabolism, and overall metabolic health.    A urine sample will 
be collected the morning of visit #2.  Urine will be collected before and following a single 
dose of blueberry or placebo containing foods on study visit #3.  Saliva will be collected on 
visits #2 and #3 for metabolomics analysis.  Supplies and instructions will be provided to 
participants. 
Blood Collection After an overnight fasting, blood will be collected from a peripheral vein by 
a trained phlebotomist at visits #2 and #3. Up to 75 ml of blood will be collected during the 
entirety of the study.  At visit #2, up to 16 ml (3.25 teaspoons) of whole blood will be 
collected following an overnight fasting. At visit #3, up to 24 ml (~1.6 tablespoons) of blood 
will be collected in the fasting state (baseline). After the initial blood draw, children will 
receive  blueberry/placebo containing products.   Participants will be free to drink water 
during this part.  Eight ml (8 ml) of blood  will be drawn 1 hour after the consumption of the 
study product while 6 ml will be drawn at 2, 3, 4, and 5 hours [Total 56 ml (24+ 32 mL) or 
3.75 tablespoons on visit #3].  We may place a saline lock and administer a slow drip of saline 
Low Polyphenol diet
Blood, stool, urine, FMD, 
start of intervention 
Daily intake of blueberry/placebo containing 
products (5 days, free living) 
Intake of blueberry/placebo containing 
product
Overnight urine collection
Blood: 0, 1, 2, 3, 4 , & 5 hour
Urine
FMD
Stool
1
Figure 2: schematic overview of study design
Study Visit
Study Visit
2
3
Study Visit
1
2
3
4
5
-1
-2
6
varies
Start of diet

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or lactate ringers at the first blood draw of visit #3 to minimize further needle sticks. If an IV 
catheter cannot be placed, and the participant agrees, standard venipuncture will be done to 
collect blood at baseline (fasting), and after 1- and 5 hours after eating the study product.   If 
blood draws need to be repeated, the total amount of blood drawn for this study will not 
exceed 75 ml.   
Plasma/serum may be used to measure levels of polyphenols, polyphenol-derived 
metabolites, and markers of cardiovascular health (e.g., lipids, glucose, etc.). Whole blood, 
plasma or serum from this study may be used in in vitro experiments to assess the immune 
response to blueberry compounds (e.g.  blueberry phenolics and blueberry volatiles), or the 
response of cells that have been either purchased (e.g., mouse or human monocyte 
macrophages) or collected in other research studies (e.g., human derived macrophages) 
where immune responses in relation to nutrition in health and disease are an outcome of 
interest. The profile of innate and adaptive immune cells in children participating in this 
study enriched products will be measured with flow cytometry. In addition, immune stress 
tests in whole blood will be done to assess immune cell responsiveness to immune stressors. 
Markers of oxidative stress (e.g., plasma protein carbonyls, malondialdehyde, 8-Hydroxy-2'-
Deoxyguanosine, and glutathione status, etc.), inflammation (C-reactive protein, erythrocyte 
sedimentation rate, and pro-inflammatory cytokines, etc.), platelet mitochondria function 
platelet activation/reactivity as well as metabolomics analyses (to characterize the 
metabolite signature associated with a dietary pattern rich in wild blueberries) may be 
conducted. Sexual hormones will be measured to complement assessment of pubertal status. 
We may conduct gene expression analyses on circulating immune cells and blood 
osteoblasts. We have previously shown that osteoblast senescence is delayed in mice 
supplemented with wild blueberries (11). Single cell RNA sequencing and/or gene 
expression tests such as GPR109A, osteocalcin, alkalin phosphoatase, NFkB, beta-catenin, 
among other. The proposed genetic analyses will not be used to diagnose disease but will be 
used as markers of bone, immune, and metabolic health. 
Endothelial function: Endothelial Function will be measured on visits #2 and #3 using 
ultrasound (GE Vivid 7 Ultrasound system with 5.0-13.0 mHz linear transducer, GE 
Healthcare, Chicago, IL, USA) to assess brachial artery flow-mediated dilation (FMD). This 

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technology uses sound waves (ultrasound) to visualize the diameter of the brachial artery. A 
baseline image of the brachial artery will be recorded at rest. Three ECG electrodes will be 
placed on the torso of the subject, such that ultrasound measurements will be captured at 
identical times during the cardiac cycle (i.e. ECG-gating of ultrasound images). A blood 
pressure cuff will be placed around the lower arm (the opposite arm that was used for 
measuring blood pressure) and the cuff will be inflated to 50 mmHg above systolic pressure 
for 5 minutes, followed by rapid deflation of the cuff. The diameter of the brachial artery will 
be measured using edge detection software (Vascular Tools, Medical Imaging Applications, 
IA, USA) from ultrasound images captured during the R wave of the cardiac cycle (via ECG-
gating) throughout the 8-minute recording protocol. The advantage of using ultrasound for 
FMD measurements is that this is a painless imaging test that uses non-invasive sound waves 
for imaging. There may be some discomfort from the blood pressure cuff since it will be 
applied tightly on the forearm. If the measurement is uncomfortable for the participant, and 
he/she requests to stop, then the procedure will be immediately aborted. FMD 
measurements will be done primarily by a registered clinical exercise physiologist with 
expertise in this procedure. However, other research staff trained and certified by Dr. 
Keshari Thakali (Director of the vascular physiology laboratory at ACNC) may conduct this 
measurement.  FMD will be measured in the fasted state in visits #2 and #3 and 1-2 hours 
after consuming a study product (visit #3 only). 
Questionnaires: A food frequency questionnaire, a medical history questionnaire, selected 
questions form the National Survey of Children’s Health, and a self-assessment questionnaire 
of pubertal status will be done at enrollment (12). Participants will have privacy to answer 
the pubertal status self-assessment questionnaire. Assessment of pubertal status is needed 
as measurements of bone metabolism are part of the present study. Puberty is known to 
dramatically affect bone turnover and metabolism (13). Physical examination for puberty 
staging is unacceptable outside the clinical setting thus self-assessment questionnaires have 
become the tool of choice in research studies.   
Study Population 
Recruitment of up to 44 children ages 11 to 12 years will be conducted by the research staff 
using IRB approved advertisements. Various advertisements will be distributed by direct 

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mail or to various locations, including but not limited to healthcare offices, health fairs, 
daycare centers, schools/universities, recreational centers, child retail stores, websites 
(ACH, ACHRI, ACNC, UAMS, and others as applicable) and churches. Also, print or digital ads 
may appear in newspapers, magazines, social media and circulars. On-hold phone messages, 
BoomText messages, screensavers, and radio/television advertisements may be used. In 
addition, ACNC research staff may contact parents who expressed an interest in our studies 
or who previously agreed to be contacted regarding future studies at ACNC. The ACNC 
research staff will educate the parents and children about the study. We will protect all 
personal information obtained during recruitment, enrollment, and testing processes. 
Inclusion Criteria 
 Ages 11 to 12 years 
 Eats ≤ 2.0 cup of fruits and ≤ 2.0 cup of vegetables per day. 
 Boys or girls 
 All ethnicities 
 All BMIs 
Exclusion Criteria 
 Known allergy to blueberries 
 Attention deficit disorder (ADD) or attention deficit hyperactivity disorder (ADHD) 
 Oppositional Defiant Disorder 
 Conduct disorder 
 Autistic spectrum disorder 
 Asthma  
 Diabetes mellitus (Type 2 and type 1) 
 neurological disease (e.g. epilepsy or seizures 
 lung diseases 
 liver disease (e.g. hepatitis) 
 kidney disease 
 Hormonal disease (e.g., hypothyroidism and growth hormone deficiency). 
 Autoimmune diseases (e.g., lupus, thyroiditis, juvenile idiopathic arthritis) 
 Bleeding disorders (e.g., hemophilia) 

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 Cancer (e.g. leukemia) 
 Chronic infections (e.g., HIV, hepatitis B, hepatitis C). 
 Pre-existing medical conditions or medications as determined by the investigators to 
affect the outcomes of interest. 
 If the participants/parents/invesitgators perceive barriers with adhering to low-
polyphenol diet. (e.g., on vegetarian diet) 
 Parent/child refusal to stop nutritional supplements (e.g., multivitamins, vitamin D, 
fish oil, probiotics, prebiotics, immune boosters, and others) 2 weeks prior to study 
visit #1. 
 Parent/child refusal to have blood drawn.  
 Parent / child unwillingness to consume the study product offered by investigators 
on study visit 3. 
 
Note: Children who had an infection that required antibiotics will be scheduled (visit #1) 
after 2 months of finalizing antibiotic treatment. Children with a viral infection such as 
diarrhea, cold or flu will be scheduled (visit #1) after 2 weeks of resolution of symptoms. 
Children will be screened (phone screening) for usage of antibiotics and occurrence of 
infections and study visits scheduled as described above. This step is necessary as immune 
responses and microbiome analyses are part of the studied outcomes and infections and 
antibiotic intake may affect these measurements  
 
Risks and Benefits 
We do not foresee any potential major adverse events associated with this protocol. Most 
procedures are minimal risk. Risk to confidentiality of personal information will be 
minimized by applying the appropriate steps to secure the collected data. Dual-Energy X-ray 
Absorptiometry (DXA) scanners use low-dose X-rays during the measurement, which may 
concern some participants/participants’ parents; however, the ionizing radiation exposure 
during a DXA scan is among the lowest found in medical procedures of this kind (about one 
tenth of a clinical chest X-ray), and is equivalent to less than two days of natural background 
radiation. There is a small risk that the participants may have an unknown allergy to the 

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blueberry / placebo enriched products. These food items (blueberry enriched only) were 
used in a sensory testing study involving 7 to 10 year old children, and no complications 
were reported (14). During the fitness test, participants may experience discomfort 
associated with exercise or the mask that is worn which is normal.  If the participant does 
not tolerate the mask and requests to stop the test, the study visit will be terminated and the 
participant will not be allowed to continue in the study. There is a minor risk that 
participants will encounter bruising and/or infection after having blood taken, however, the 
use of well-established blood draw techniques, and antiseptic procedures, by trained 
phlebotomists, will ensure minimal risk. Participants may also experience sickness or 
dizziness associated with blood draw and fasted state. However, this and any other 
procedures can be aborted at any time at the request of the participant.  Genetic testing may 
present a unique risk to human research subjects.  These involve medical, psychosocial and 
loss of confidentiality. To minimize these risks, participate names will be removed from the 
samples, and kept in secure and badge access only laboratories. In addition, the proposed 
tests are for research purposes only, that is, they will not be used as diagnostic tests for any 
disease or illness and will not be placed in medical records or shared with treating 
physicians. 
In the event of an adverse event or unanticipated problems, this will be reported to 
the study PIs, the IRB, and the study sponsor in accordance with IRB Policy 10.2. 
There will be no direct benefits to the study participants; however, knowledge gained 
from the study could potentially benefit patients in the future. 
 
Data Handling and Recordkeeping 
All investigators and study staff will complete and maintain appropriate CITI 
training. All data and communications will be recorded in either electronic or paper copy 
standardized case report forms. Survey and questionnaire data will be entered directly into 
REDCap or Remark Web Survey Professional on study iPads when possible by participants, 
with paper versions of all forms to be used as back-up in the instance that iPads are 
unavailable. Remark Web Survey Professional will be used with participants ID only and 
therefore no identifiers will be available on the Remark Web Survey Professional. All data 

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and communications will be recorded in standardized case report forms or directly in the 
ACNC clinical relational database (MS Access, Redmond, CA). Data will be collected and 
managed using the ACNC clinical relational database (MS Access, Redmond, CA). The data 
integrity will be password protected. Data analyses will be performed from queries which 
will not change and/or modify the original data from the tables. This database will be 
housed on a shared drive backed up nightly off-site Monday – Friday. Access to our server 
is password protected, as is access to the study database(s). Only faculty and staff who have 
had appropriate CITI training have access to study database(s) with participant identifiers.  
The Principal Investigator will carefully monitor study procedures to protect the 
safety of research subjects, the quality of the data and the integrity of the study. All study 
subject material will be assigned a unique identifying code or number (e.g. participant ID). 
The key to the code will be kept in a shared network folder with access granted only to 
study staff who have had appropriate CITI training. Only study investigators/staff will have 
access to the code and information that identifies the subject in this study. At the 
conclusion of the study, the data will be stored in the database and/or secure server. 
Specimen Handling and Storage  
During the consent process, parents will be given the option to allow banking of 
specimens obtained from their child over the course of the study. Participants’ names will be 
removed from specimens. Specimens will be stored indefinitely for potential use in future 
research studies that are within the scope of the current one. Urine, stool, and blood samples 
will be stored in freezers in locked laboratories at Arkansas Children’s Nutrition Center and 
Arkansas Children’s Research Institute. Participants may choose to withdraw permission for 
banking at any time they decide. A letter or e-mail addressed to the principal investigators 
expressing their desire to withdraw their consent would suffice. Consent for banking will be 
separate from consent to participate in the current study. Choosing not to have their 
specimens stored for future analyses will not affect their eligibility for this study. 
Participants may call the investigators or research staff to obtain more information if needed. 
 
Data Analysis 

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Statistical analysis: Based on means and standard deviations observed in previous similar 
studies, our primary outcome will have in excess of 80% power to detect statistical 
significance among the study groups testing at the 5% level of significance.  This calculation 
assumes that we have at least n=9 subjects in each of our four combinations of gender and 
blueberry status. This being the case, we propose to recruit n=11 subjects in each 
combination of gender and blueberry status (n=44 total, 22 per study arm) to compensate 
for a reasonable level of dropout that may occur.  Power and sample size calculations were 
performed by SAS 9.4 Proc Power.  David Williams, PhD, ACNC statistician will be in charged 
randomization process. Prior to analyses, data will be checked for potential outliers and 
aberrant measurements. Additionally, the data will be also checked for conformity with 
distributional assumptions of the proposed statistical models. Analysis of variance (ANOVA) 
or its non-parametric variants will be used to assess differences among gender and 
blueberry diet combinations. Significance will be declared at the 5 percent level of 
significance. To learn whether peak VO2 indeed modifies the effect of WBB supplementation, 
the potential modifier will be added into the ANOVA thus making an analysis of covariance 
(ANCOVA). The slope of the modifier will be allowed to depend on WBB status; that is, there 
will be a slope for the placebo group and one for the WBB group. 
Reimbursement 
The compensation per visit are $50 for visit #1, $50 for visit #2, and $175 for visit #3 (Total 
$275). If the participant decides not to complete the study, is unwilling, or unable to 
complete an assessment, a $25 gift card or check will be provided.   
In the event that blood draws or other measurements need to be repeated and re-
scheduled due to technical problems (but not participant refusal to blood draw), an 
additional $50 gift card or check will be provided.  
 
Ethical Considerations 
 
This study will be conducted in accordance with all applicable government 
regulations and University of Arkansas for Medical Sciences (UAMS) research policies and 
procedures.  This protocol and any amendments will be submitted and approved by the 
UAMS Institutional Review Board (IRB) to conduct the study.  Formal consent and assent, 

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using the IRB-approved consent/assent form, will be obtained before the subject is 
submitted to any study procedure.  Parents may also be contacted before study visit (s) to 
confirm appointments and ensure eligibility.  All parents and children for this study will be 
provided with a consent/assent form describing this study and providing sufficient 
information in language suitable for them to make an informed decision about their 
participation in this study. A copy of the consent/assent may be mailed or e-mailed to them 
for information after successful telephone screening. Upon arrival to the ACNC, the 
consent/assent process will take place in a quiet and private room, and children and 
parents may take as much time as needed to make a decision about their 
participation.  Participation privacy will be maintained and questions regarding 
participation will be answered.  No coercion or undue influence will be used in the consent 
or assent process. Potential participants will be asked if their information can be saved for 
consideration for future studies and their responses will be documented. The person 
obtaining consent and assent will outline the risks, benefits, and requirements of the study 
as well as review all study procedures and the informed consent/assent documents with 
the child and parent.  If eligible and interested in enrollment, informed consent will be 
obtained from the parent and documented, and assent will be obtained from the child and 
documented. A copy of the signed consent and assent will be given to the participant, and 
the informed consent process will be documented in each subject’s research record.   
 
Dissemination of Data 
Results of this study may be used for presentations, posters, or publications. The 
publications will not contain any identifiable information that could be linked to a 
participant.  Parent may also receive a report containing some of his/her child’s results. The 
study will be listed on clinicaltrials.gov in accordance with (journal or FDA) requirements. 
 
 

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