The Effects of Watermelon Juice Supplementation on Postprandial Vascular Endothelial Function and Blood Flow During Hyperglycemia: A Pilot Study
Phase: NA
Conditions: Endothelial Dysfunction
Sponsor: Pennington Biomedical Research Center

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Protocol Title: The Effects of Watermelon Juice Supplementation on Postprandial 
Vascular Endothelial Function and Blood Flow During Hyperglycemia: A Pilot Study 
 
 
PI Name: Timothy Allerton Ph.D., LCEP 
 
Sub-Investigator’s Name(s): 
Brian Irving Ph.D. 
Neil Johannsen Ph.D. 
Guillaume Spielmann Ph.D. 
Jack Losso Ph.D.  
 
Medical Investigator: Daniel Hsia, MD 
 
Protocol Version Date: 04/29/19 
 
 
Objectives 
The objective of this pilot study is to determine the potential for watermelon juice to attenuate 
the reduction postprandial endothelial function and skeletal muscle microvascular blood flow 
(MVBF) experienced during hyperglycemia. 
 
We will attempt to answer the following hypotheses: 
Hypothesis 1: Watermelon juice supplementation will attenuate the reduction in endothelial 
dysfunction and microvascular blood flow during an oral glucose challenge. 
 
Hypothesis 2: Watermelon juice will increase L-arginine bioavailability during hyperglycemia and 
correlate with improved vascular response. 
 
Exploratory Aim: The postprandial period is defined by increased sympathetic nervous system 
activity (vasoconstriction) and NO mediated vasodilation. Heart rate variability (HRV) is a 
measurement of the balance of parasympathetic to sympathetic activity.  We will measure HRV 
during the oral glucose challenge to interrogate the possibility that watermelon juice can 
modulate the balance of blood vessel constriction and relaxation during an oral glucose 
challenge.   
 
Background 
Cardiovascular disease is the primary cause of death in people with obesity and type 2 diabetes 
(1, 2). Postprandial hyperglycemia, hyperlipidemia, and endothelial dysfunction are strong 
predictors of future CVD events and death (3, 4). Nitric oxide (NO) mediated vasodilation is a 
critical component of blood pressure regulation, endothelial function, and insulin-mediated 
glucose disposal (5, 6).  NO is also essential to insulin-mediated vasodilation of large and 
microvascular blood vessels in response to a nutrient challenge (mixed-meal, glucose, fat).  The 
capacity of insulin to increase vasodilation and postprandial microvascular blood flow (MVBF) 
accounts for 40-50% of insulin-stimulated glucose disposal (5, 7). Strategies to increase the 
vasodilatory actions of insulin would therefore make a significant impact on vascular and 
metabolic health.  
 
Several studies have documented reduced NO synthesis in obesity and type 2 diabetes(8-10). 
In endothelial cells, NO is synthesized from L -arginine (precursor) by endothelial-nitric oxide 

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synthase (eNOS) generating NO and L –citrulline (products)(11) [adjacent figure].  
Supplemental L-arginine, at high doses (9-24 g), has been shown to improve postprandial 
endothelial function in response to a high-fat meal in those with low plasma arginine levels but 
did not improve postprandial blood flow (12).  However, oral L-arginine supplementation is 
subject to a high degree of first-pass extraction and elevates arginase enzyme activity. 
Furthermore, chronic L-arginine supplementation has been shown, in some cases, to be 
ineffective and unsafe for those with cardiovascular disease (13, 14).  
 
The amino substrate for L-arginine, L-citrulline, is more effective at increases circulating L-
arginine than L-arginine supplementation itself (15-17).   L-citrulline is rapidly acted on by 
cytosolic arginosuccinate synthase and converted into arginosuccinate which is then converted 
into L -arginine by arginosuccinate lyase. L-citrulline or watermelon (the most abundant dietary 
source of L-citrulline) has been shown to be effective at improving some reactive cardiovascular 
measurements in normo, pre-, and hypertensive participants (18-20). Preclinical trials have 
demonstrated potential for L-citrulline to protect against endothelial damage when consuming a 
high fat/cholesterol diet (21). 
 
Several studies exhibit that hyperglycemia, even in healthy populations, impairs endothelial 
function and microvascular blood flow by reducing NO bioavailability (22-25).  Increasing the 
bioavailability and action of NO  by increasing L-arginine levels or reducing the ratio of 
asymmetric dymethylarginine (ADMA) to L-arginine has the potential to confer protective effects 
against hyperglycemia-induced endothelial dysfunction.  Based on the available data, 
supplementation with L-citrulline has greater potential, than oral L-arginine supplementation, to 
increase systemic L-arginine bioavailability.  Utilizing a natural food source high in L-citrulline 
(watermelon juice) to improve the postprandial vascular response to hyperglycemia has not 
been studied to date. The purpose of this pilot study is to determine the potential for 
watermelon juice to attenuate the reduction in postprandial endothelial function 
associated with reduced skeletal muscle microvascular blood flow (MVBF) during 
hyperglycemia. 
Inclusion and Exclusion Criteria 
Study Population. We will recruit, randomize, and study up to 20 young (18-40 years 
old), healthy (no metabolic disease), and participants with a normal BMI (18-29.9 kg/m2) 
from the LSU community.  
 
Inclusion and Exclusion Criteria 
 
Inclusion Criteria.  
1. Capable and willing to give written informed 
consent and understand inclusion and exclusion 
criteria.  
2. 18-40 years of age  
3. BMI between 18-29.9 kg/m2 
4. Willing to allow researchers to draw blood and 
conduct imaging (DXA) for research purposes.  
 
Exclusion Criteria.  
1. Evidence or self-reported history of type 1 or 2 diabetes mellitus 
2. Self-reported family history of type 2 diabetes (first degree relative with type 2 
diabetes) 
3. BMI > 29.9 kg/m2 

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4. Evidence or self-report history of deep vein thrombosis, pulmonary embolism, 
cardiovascular, peripheral vascular, cerebral vascular, pulmonary, or renal 
disease 
5. Allergy to watermelon 
6. Use of medication known to influence study outcomes, such as: 
a. Insulin 
b. Anti-diabetics (metaformin) 
c. Corticosteroids 
d. Beta-blockers 
e. Anti-coagulants 
7. Use of supplements known to influence study outcomes, such as; 
a. Beta-alanine 
b. L-arginine 
c. L-citrulline 
8. Active smoking 
 
 
Number of Subjects 
N=20 
 
 
Recruitment Methods 
We will recruit primarily on LSU campus via word of mouth and flyers.  
 
Study Timelines 
 
Based on the project notification date of March 15th, 2019 we plan to proceed with study 
recruitment in April of 2019 with a study approximate study start at the end of May 2019. The 
total study commitment for each participant is 42 days. We anticipate completing the study in 
the early fall (September/October) of 2019.  Given these target dates are met we plan to 
complete the preliminary analysis by December 2019.  
 
Study Endpoints 
The primary endpoints for the study are changes in postprandial flow-mediated dilation 
and microvascular blood flow. Secondary endpoints will include plasma biomarkers of 
nitric oxide active and oxidative stress. We will also explore changes in heart rate 
variability as a secondary outcome.  
 
For the oral glucose tolerance test participants arriving with fasting blood glucose >100 
mg/dl will not proceed with testing and will be referred to the student health center for 
follow up after consulting with the study MI. Any blood glucose > 200 mg/dl during the 
OGTT will result in test termination and referral to the student health center after 
consulting with study MI.  
 
Procedures Involved 

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There will be 1 screening visit, and 2 outpatient visits. Participants will consume watermelon 
juice or placebo for 2 weeks. The washout period will last 2 weeks prior to the cross-over to the 
opposite condition. Participant visits will be conducted at the Vascular and Resting Metabolism 
Lab located in the Department of Kinesiology at LSU.  
 
Screening Visit  
Participants will undergo consenting and fasting blood draws to measure glucose, lipids, and 
CBC. Vital signs (blood pressure, body weight, body composition (DXA), heart rate, etc) will be 
measured. For DXA procedure participants will undergo a whole body scan lasting 
approximately 10 minutes. Participants will remove all metal objects from their body and lie 
down on the table. The legs will be secured together using two Velcro straps. The participant 
will be instructed to remain completely still during the scan. Randomization will be performed to 
allocate the study participant for their initial group assignment to either the watermelon juice 
group or placebo. Group assignments will be generated by randomization software 
(http://www.graphpad.com/quickcalcs/index.cfm) third-party faculty member not 
associated with the study.   
Daily Juice Drop-in visits 
Since the required supplementation duration is 14 days participants will be provided 
with 2 days (Saturday and Sunday) worth of watermelon juice or placebo to account for 
days when the LSU AG Center will not be open. Participants will also be provided with 
juice or placebo for anticipated instances where they cannot be on campus. In order to 
monitor compliance participants will be asked to return the juice container for the days 
where juice was consumed off-premises.  
 
 
Visit 2 and 3: Oral Glucose Challenge – Postprandial 
FMD and MVBF 
Participants will arrive in morning at 6:00am fasted for 10-
hours. Body composition will be measured by DXA. Next, 
participants will rest for 30 minutes in the supine positing 
while wearing a heart rate monitor (Zephyr, Bioharness) 
to measure heart rate and heart rate variability. The 
resting metabolic rate will be measured via indirect 
calorimetry for 20 minutes. Then fasting measures of 
blood glucose, FMD (ultrasound), and MVBF (near-
infrared spectroscopy) will be taken followed by ingestion 
of 75 g of glucose (glucola). An IV line will be placed in the participant's arm vein for blood 
draw purposes and will remain there throughout the testing.  A blood sample will be 
drawn, and then the participant will drink a sugar solution consisting of 75 grams of 
glucose.  Blood will be drawn at specific times after you consume the drink.  Each blood 
sample will be about 1 tablespoon. (6 tablespoons total for the test). During the IV 
procedure, a small amount of the participant’s own blood (less than 1 teaspoon) 
will immediately be returned into your vein through the IV after each specimen is 
collected.   Blood will be drawn at minutes 15, 30, 60, 90, and 120 minutes. Postprandial 
measurements of FMD and MVBF will be taken at 30, 60, 90, and 120 minutes. 
 
The oral glucose challenge will be conducted by Drs. Allerton, Johannsen, Spielmann, and 
Irving under the medical direction of Dr. Hsia.  
 
Research Methods  
 
 
 
 
 
 
 
 
 
 
Enrollment
Randomization
WMJ
Placebo
Follow-up 1
Washout 2 
weeks/crossover
Figure 1. Experimental Design
2 weeks
Placebo
WMJ
2 weeks
Follow-up 2

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To measure postprandial FMD and MVBF we will use a standard oral glucose tolerance test 
format with periodic measurement of substrate oxidation (indirect calorimetry).   
 
Flow Mediated Dilation 
FMD will be measured according to standard procedures supported by the American Heart 
Association. A blood pressure occlusion cuff will be placed on the forearm distal to the 
ultrasound probe in preparation for measurement.  The brachial artery of the right arm will be 
identified and scanned using B-mode ultrasound after a 10-minute resting period. Continuous 
cross-sectional images will be recorded for 3-minutes at rest, during cuff inflation (250 mmHg for 
5 minutes) and flowing release of cuff inflation. Fasting measurement will take place -30-
mintutes, whereas, postprandial measurement will take place +30, +60, +90, and +120 minutes.  
Vascular images will be acquired using a 10mHz linear array transducer (8L-RS).  
FMD will be calculated as follows: 
 
Raw values for vessel diameter and shear stress will also be collected and reported.   
 
Microvascular Blood Flow-Near Infrared Spectroscopy (NIRS) 
The NIRS optodes (Oxymon MKIII, Artinis Medical Systems) will be placed longitudinally on the 
flexor digitalis superficialis (forearm).  The optodes will be secured in place with two-sided 
adhesive tape and further anchored in place with additional medical tape.  A blood pressure cuff 
(Hokanson, Bellevue WA) will be placed proximal to the optodes, distal to the FMD ultrasound 
probe and connected to a rapid cuff inflation system (Hokanson E20, Bellevue WA) controlled 
using a custom-built control system and Labview Software algorithms (developed by Pedro J 
Chacon Dominguez, Jin-Woo Choi, and Brian A Irving).  NIRS signals for oxygenated 
(O2Hb/O2Mb), deoxygenated (HHb/HMb), and total (tHb/tMb) hemoglobin/myoglobin will be 
continuously monitored (29). 
 
 
 
 
 
 
 
Table 1.  
 
Screening 
Visit 
2 weeks (WMJ or PLA) 
Study Visit 1 
Washout- 2 weeks 
2 weeks WMJ or PLA 
Study Visit 2  
Informed Consent 
X 
 
 
Randomization 
X 
 
 
Height/Weight/BMI 
X 
 
 
Vital Signs (blood 
pressure, heart rate)
X 
 
 
Questionnaires 
X 
 
 
DXA 
x 
x 
x 
Oral Glucose 
Challenge 
 
x 
x 
RMR 
 
x 
x 
FMD/MVBF 
 
x 
x 
Blood Draws 
x 
x 
x 
Daily Trips to Drink 
Juice or Placebo 
 
 
x 
 
 
x 

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Resting muscle microvascular blood flow will be measured -30 minutes in triplicate using 
venous occlusions by inflating the blood pressure cuff to (50-60 mmHg) for 20 seconds with at 
least 120 seconds between each measurement (29).  The same procedure will be conducted 
+30, +60, +90, and +120 minutes post-glucose challenge. We will measure blood flow by 
calculating the slope of the linear increase total hemoglobin (tHb): BF= (((tHb x 60)/ ((([Hb] 
x1000)/4))x1000/10 in ml-1min100ml-1 (29). *NOTE: FMD and MVBF measurement times will 
be staggered 3 minutes after blood draws to avoid confounding factors.  
 
Lab Samples: Measurement of glucose, insulin and free fatty acids will be measured as a part 
of the basic metabolic profile for the oral glucose challenge. Fasting and postprandial 
measurements of arginine, citrulline, and ADMA will be measured to compare the impact of 
watermelon juice consumption on postprandial amino acid concentrations. Plasma markers of 
endothelial function (vascular adhesion molecule-1 (VCAM-1), E-selectin, plasminogen activator 
inhibitor-1 (PAI-1), and CRP) and oxidative stress (peroxinitrite, reduced gluthatione, oxidized 
LDL) will also be measured.   
 
Figure 2. 
 
 
 
 
 
 
Risks Associated with Procedures 
 
Blood Draws:  There is the possibility of infection and/or pain and bruising at the vein 
on the arm where the needle is inserted.  Aseptic (sterile) technique and trained 
personnel minimize these risks 
 
OGTT:   There is a possibility of pain, bruising, or infection at the site of the needle 
insertion for the IV line.  Trained personnel minimize this risk.  The drink may make 
cause nausea, vomiting, abdominal bloating, or a headache.   
 
Whole Body Scan GE iDXA  (Dual energy x-ray absorptiometry) 
The amount of radiation used for this procedure is very small.  The radiation dose for 
this scan is equivalent to the radiation that one would naturally exposed to in the 
environment in less than one day.  Scans will not be performed on any subject who is 
pregnant.  A urine pregnancy test will be performed within 72 hours before the scan on 
females of child-bearing potential.     
 

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Muscle Blood Flow and Metabolism Measurements:  Inflation of the blood pressure 
cuff around the arm may cause some discomfort and pain during the test.  The 
temporary numbness and tingling are similar to the sensation of having the hand “fall 
asleep”.  If the discomfort is too severe,the participant may stop the test by notifying the 
technician to stop and the test will be immediately terminated.  There is no known risk 
from the use of skin folds and/or ultrasound to measure adipose tissue thickness. 
  
Power analysis. 
To detect a 30% difference in FMD or MVBF (power =0.80, =0.05) we will require 18 
participants. Assuming a 10% drop out rate this will require the need to enroll 20 participants.  
 
Data and Specimen Management 
A mixed model with repeated measures will be employed to assess pre- oral glucose vs. 
postprandial changes in FMD, MVBF, and plasma markers. 
 
Participants will be provided an identification code not linked to any personal 
information. Study data will be input into an excel spreadsheet and saved to the 
department of kinesiology drive and password protected.  Only the study PI and co-
investigators will have access to the study files. Paper files will be locked in a secure 
filling cabinet with access only provide to study personnel. Likewise, at LSU, to preserve 
confidentiality and data integrity, all data collection forms obtained will be secured in 
locked filing cabinets, or on password protected computers. Access to the primary data 
collection forms will be under the control of the PI and approved study personnel. 
Consistent with good clinical practice we will keep hard copy data records for a 
minimum of 3 years at which time they may be destroyed or kept indefinitely. 
 
Biospecimens will be temporarily stored in secured -80 freezer at LSU kinesiology then 
will be transported by the study PI (Tim Allerton) in the following manner.  
 
Plasma and serum samples will be transferred from Louisiana State University 
department of kinesiology to Pennington Biomedical Research Center. Samples will be 
in clearly labeled, leak-proof primary containers placed in a biohazard labeled, leak-
proof secondary container containing absorbent material sufficient to retain the total 
sample volume.  The packaged samples will be placed on dry ice in a Styrofoam 
transport vessel and transported in a non-passenger compartment of Dr. Allerton’s 
vehicle with a printed sample log or other documentation describing the samples being 
transported. Dr. Allerton will maintain a biohazard spill kit in the vehicle. 
 
Provisions to Monitor the Data to Ensure the Safety of Subjects 
This Human Subjects Research meets the definition of a Clinical Trial. This study does 
not involve major risk to subjects. Efforts to minimize the potential risks of the 
assessment methods and outcome variables include frequent monitoring by the 
investigators to assure that no volunteer suffers any adverse effects from participating in 
the research. 
 
Safety Monitoring. In this study, an adverse event or experience is defined as any health-
related unfavorable or unintended medical occurrence that happens throughout study 
participation. Examples of adverse events include but are not limited to the following: 

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 A clinically significant laboratory or clinical test result. 
 An event that results in missing a study visit. 
 An event that requires a visit to a physician. 
 An event that occurs as a result of a study procedure. 
 
 
Withdrawal of Subjects 
Subject could be withdrawn at any time during the study by Dr. Allerton (PI) or Dr. Hsia 
(study MI) on the grounds that removal from the study would be in the best interest of 
the subject. Non-compliance to the study protocol could also result in removal from the 
study. The IRB and or the sponsor may terminate the study. Additionally, study subject 
may withdraw voluntarily from the study themselves.  
 
Potential Benefits to Subjects 
There is no direct known benefit for subjects participating in this study.  
 
Vulnerable Populations 
No vulnerable population will be studied in this trial.  
 
 
Sharing of Results with Subjects 
 
The results of the tests done in the study will be analyzed and published in a medical 
journal. The results will be summarized and shared with the study subjects. After the 
study, individual results will be made available to the study subjects and, at their 
request, with their primary physician 
 
Setting 
The study screening visit, and visit 1 and visit 2 will be conducted in the department of 
kinesiology at LSU Vascular and Resting Metabolism Laboratory. Dr. Tim Allerton will 
serve as study PI, with Drs. Neil Johannsen, Brian, Irving, and Guillaume Speilmann will 
serve as study Co-Investigators. Dr. Daniel Hsia will provide medical oversight at study 
MI>  
 
Resources Available 
Vascular and Resting Metabolism Laboratory (VRML) (500 ft2) is OSHA approved for 
blood work and consists of five rooms (office, work room, metabolic testing suite, and 
heat chamber).  The VRML is equipped with a near-infrared spectrometer (Oxymon Mk 
III NIRS system, Artinis), a rapid cuff inflation system (E20, Hokanson), portable 
vascular ultrasound (LOGIC e, GE), Brachial Analyzer for Research Software (Vascular 
Research Tools, MIA-LLC), SphygmoCor (AtCor Medical), Caposcope, Laser Doppler 
(PeriFlux 5000, Perimed), data acquisition system (MP150, Biopac), biochemistry 
analyzer (GL5, Anolox), cholesterol analyzer (Cholestech LDX, Alere), electrolyte 
analyzer (EasyLyte, Medica), portable lactate analyzers (Lactate Pro), centrifuge, vapor 
pressure osmometer (Vapro, Wescor), and a hygrometer (Dew Point Mirror 473). 
 
 
Prior Approvals 

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This study will be supported by that National Watermelon Promotional Board.  
 
Compensation 
Participants are eligible to be paid $75.00 for completion of the study.  
 
Participants will receive $25 for completing at least one study visit paid upon notification 
of drop out of study. Participants will not be compensated for completing the screening 
visit.  
 
Provisions to Protect the Privacy Interests of Subjects 
Participants will review the consent form will study personnel in a private room. The 
signed consent form will be stored in a secure file cabinet. The subject will receive a 
copy of the study consent. We will explain to the subject that the results will be 
published in a medical journal, but subjects will not be identified.  
 
Compensation for Research-Related Injury 
No form of compensation for medical treatment or for other damages (i.e., lost wages, 
time lost from work, etc.) is available from the Pennington Biomedical Research Center 
or LSU. In the event of injury or medical illness resulting from the research procedures 
in which a subject participates in, the subject will be referred to a treatment facility. 
Medical treatment may be provided at the subject’s expense or at the expense of 
his/her health care insurer (e.g., Medicare, Medicaid, Blue Cross-Blue Shield, Dental 
Insurer, etc.) which may or may not provide coverage. The Pennington Biomedical 
Research Center is a research facility and provides medical treatment only as part of 
research protocols. Should a subject require ongoing medical treatments, they must be 
provided by community physicians and hospitals. 
 
Economic Burden to Subjects 
We do not anticipate any economic burden as a part of this study.  
 
Consent Process 
Written informed consent will be obtained from the subjects taking part in this study. The 
consent process will be initiated prior to screening by Drs. Allerton, Irving, Spielmann, 
Johannsen, or IRB approved personnel but the process will continue throughout the 
study and subjects will be encouraged to ask any questions they may 
have. If subjects wish to take the consent home for further reflection or to discuss with 
their family or counselors, this will be permitted.