Impacts of a Combined Berry Extract Supplement on Endothelial Function, Blood Pressure, Oxygen Utility Capacity, and Fatigue Index in Healthy Young Adults
Phase: NA
Conditions: Healthy
Sponsor: University of Nebraska

Impacts of a combined berry extract supplement on vascular function and oxygen utility capacity in 
young adults: full study protocol and statistical analysis plan 
NCT#04312022 
10-04-2018 
  
 
 

Study protocol  
Participants 
Healthy adults (n = 18, age 19-35, 9 males and 9 females) who were recreationally active volunteered to 
participate in this study. Exclusion criteria included 1) any cardiovascular, neurological, metabolic, 
respiratory, or renal diseases, 2) any musculoskeletal conditions or injuries, 3) presence or history of 
stomach ulcers, 4) prescribed medications or over-the-counter medications, 5) pregnant, trying to 
become pregnant, or breast-feeding women, 6) any history of smoking/current smoker, and 7) any 
allergies to fruits or vegetables. The procedures used in this study were approved by the Institutional 
Review Board at the University of Nebraska Medical Center and carried out in accordance with the 
Declaration of Helsinki. All participants gave their written informed consent prior to study enrollment, 
during which the experimental procedures, potential risks, and potential benefits were explained. This 
study was registered with https://clinicaltrials.gov/ (NCT04312022). 
 
Study design 
A randomized, double-blind, placebo-controlled, crossover study design with a 2-week washout period 
was used. After study enrollment, participants were randomly assigned to either the BE supplement 
group or the placebo control group. All data collections were performed at the same time of day (± 1h) 
after an overnight fast, and participants were asked to abstain from caffeine, alcohol, high intake of 
antioxidant-rich foods or supplements, and excessive exercise for at least 48 hours prior to their visits. 
Participants were also informed to not change their dietary habits during the study period. Descriptive 
measurements of height, weight, BMI, body composition, and hand grip strength were taken at both BE 
and placebo visits. Baseline measurements of RHR, BP, blood sampling for TAC, endothelial function, 
skeletal muscle oxygen utility capacity during exercise, and muscular fatigue index were assessed. 
Participants then consumed either the BE or placebo, and all baseline measures were repeated as 
experimental measures 1 hour after BE or placebo intake. A 1-hour digestion period was chosen because 
anthocyanins have been shown to reach peak levels in the blood within 1-hour post consumption1,2. 
After 2 weeks each participant returned for a second visit and the same protocol was repeated with the 
intervention (BE or placebo) they did not receive in the first visit. All women (n = 9) were tested during 
the early-to-mid follicular (days 1 – 10) and late-luteal phases (> day 19) of the menstrual cycle in order 
to avoid confounding effects of endogenous estrogen on autonomic function3. 
 
Supplementation 
The antioxidant supplement used in this study contained 3 natural plant-based ingredients including 1) 
hawthorn berry extract, derived from the hawthorn berry (Crataegus spp.), 2) tart cherry powder 
(Prunus cerasus), a rich source of anthocyanins, and 3) bromelain, a mixture of proteases obtained from 
the stems and immature fruits of pineapples. The BE supplement consisted of 2 capsules with a total of 
465mg, 480mg, and 400mg of hawthorn extract, tart cherry powder, and bromelain, respectively 
(CardioEffects, Fitfully LLC, Omaha, NE, USA). This is similar to other doses used in previous studies and 
has been shown to have no adverse side effects4,5. The anthocyanin content for this supplement was 
provided by the company, in which two capsules contain ~68 mg of anthocyanins. The separate 

components include ~67 mg from tart cherry and ~2000 ng from hawthorn berry, which is consistent 
with previous studies4,6. The tart cherry anthocyanin content is similar to ~31 mL of tart cherry juice4 
while the hawthorn berry anthocyanin content is similar to ~1g of dried hawthorn berries6. Bromelain is 
primarily extracted from pineapple stems7, and therefore bromelain is more often given in supplement 
form than by natural consumption. All capsules used in the present study came from the same batch 
from the manufacturer. The placebo consisted of 2 tapioca powder capsules that were identical in size 
and appearance to the BE supplement and did not possess any antioxidant properties. The capsules were 
given to the participant by a lab member not directly involved in the study measurements or analyses. 
 
Anthropometrics 
A standard stadiometer was used to measure height to the nearest 0.1 cm. Body mass was measured to 
the nearest 0.1 kg using a standard scale. Body mass index (BMI) was calculated as the body mass 
divided by the square of height (kg/m2). Body fat percentage was quantified using handheld bioelectrical 
impedance analysis (BIA) (Omron, model HBF-306C, Omron Healthcare, Inc., Lake Forest, IL). Body fat 
percentage was measured in duplicate, and the average of the 2 was recorded as the body fat 
percentage. 
 
Resting heart rate and blood pressure 
RHR and BP were assessed before and after BE and placebo intake. Participants rested in a seated 
position in a quiet room for 5 minutes and were informed not to talk or move during this time. RHR, 
systolic BP, and diastolic BP were measured using an automated sphygmomanometer (Omrom Blood 
Pressure Monitor BP786N, Omron Healthcare, Inc., Lake Forest, IL) in duplicate. The two measurements 
of the RHR and BP were averaged and recorded as the resting values. 
 
Blood sampling 
Blood samples were collected from an antecubital vein by a trained phlebotomist using EDTA tubes 
before and after BE and placebo intake. Samples were centrifuged at 3500 rpm for 10 minutes at 4°C. 
Plasma samples were stored at -30°C for later analysis of total antioxidant capacity (TAC). TAC was 
assessed using a commercially available Total Antioxidant Capacity Assay Kit (CAT#: ab65329, Abcam, 
Cambridge, UK) according to the manufacturer’s instructions. After incubating samples at room 
temperature (23°C), absorbances were measured at 570nm using a microplate reader. The average intra-
assay and inter-assay coefficient of variation for TAC were 6.3% and 2.9%, respectively. 
 
Endothelial function 
Flow-mediated dilation (FMD) of the brachial artery was used to assess endothelial function before and 
after BE and placebo intake. FMD is an endothelium-dependent assessment that facilitates brachial 
artery relaxation in response to an increase in shear stress. FMD was assessed using a Terason uSmart 
3300 Doppler ultrasound system (Terason Division Teratech Corporation, Burlington, MA) and EKG trigger 

monitor (7700 Series Trigger Monitor, IvyBiomedical Systems, Inc., Branford, CT). The ultrasound probe 
was used to locate the brachial artery on the participant’s right arm, and a rapid-inflation cuff (E20 Rapid 
Cuff, D.E. Hokanson, Bellevue, WA) was placed on the forearm distal to the ultrasound probe. A baseline 
resting brachial artery diameter was recorded for 5 minutes using an image capturing system (Vascular 
Imager, Vascular Research Tools 6, Medical Imaging Applications, Coralville, IA). The cuff was then 
inflated to 250 mmHg for 5 minutes. The cuff was released, and the reactive hyperemic response of the 
artery was recorded for 5 minutes using the ultrasound and image capturing system. The baseline resting 
diameter and post-hyperemic stimulus were analyzed using automated edge detection software 
(Brachial Analyzer, Vascular Research Tools 6, Medical Imaging Applications, Coralville, IA). The most 
stable 30-60 seconds of the baseline artery diameter measurement, including at least 10 cardiac cycles, 
was averaged as the resting diameter 8. 
 
Skeletal muscle oxygen utility capacity 
Skeletal muscle oxygen utility capacity during single leg extension exercise was assessed before and after 
BE and placebo intake. Oxygenation utility capacity of the vastus lateralis was measured during leg 
extension exercise with a commercially available NIRS system (Artinis PortaMon, Einsteinweg, The 
Netherlands). The PortaMon emits near-infrared wavelengths of 850 and 764 nm and has a detection 
probe to measure returning signals, and data were recorded continuously at 10 Hz to quantify tissue 
saturation index (StO2, %) and concentrations of both oxygenated hemoglobin ([O2Hb], a.u.) and 
deoxygenated hemoglobin ([HHb], a.u.). 
To determine single leg extension strength, a 1-repetition maximum (1RM) test was performed using the 
participant’s dominant leg. Participants were familiarized with the leg extension technique before the 
1RM measurement, which was achieved within 3 attempts. The 1RM was considered the highest weight 
that could be lifted in good form through a full range of motion. The PortaMon was secured with a 
commercially available, double-sided adhesive at one- third of the distance from the lateral femoral 
epicondyle and the greater trochanter, and the device was adjusted to be on the belly of the vastus 
lateralis muscle9. The device was wrapped in black, light-absorbing cloth to reduce extraneous light that 
may affect the signals. Participants were then asked to perform 15 repetitions at 60% of their 1RM while 
NIRS data were recorded continuously throughout the exercise protocol. 
 
Fatigability index 
Fatigability index was quantified during Isokinetic contraction of the participant’s dominant leg before 
and after BE and placebo intake using HUMAC NORM Isokinetic Dynamometer (CSMi Solutions, 
Stoughton, MA). The participants were seated upright with the axis of rotation of the dynamometer arm 
oriented with the axis of rotation of the participant’s dominant knee. Belts and straps were used to 
secure the participant to the dynamometer, and participants were instructed to fully extend and flex 
their knee and to work to their maximal capacity during the leg extension exercise. Before the test, there 
were 4 familiarization repetitions at the testing resistance. The testing consisted of a mild resistance at 
240°/sec to induce and measure muscular fatigue. The software (HUMAC 2015, v.15.000.0103) reported 
the data of fatigue index, which was calculated using the percentage peak torque differences that were 

found on the first and final 5 repetitions of the exercise bout (fatigue index = [initial peak torque – final 
peak torque]/initial peak torque * 100). The data obtained from the analysis software allowed for the 
assessment of changes in numbers relative to the participant baseline measurements. 
 
Statistical analysis 
A Shapiro-Wilk’s test was used to determine normal distribution of the data. Independent t-tests were 
used for evaluating baseline characteristics at the BE and placebo visits. Dependent variables were 
assessed suing a 2 x 2 repeated measures analysis of variance (ANOVA) [group (BE and placebo) x time 
(before and after supplement intake)] to determine differences between pre- and post-BE and placebo 
intake. If a significant effect was noted, paired t-tests were used for post hoc comparisons. All statistical 
analyses were performed with SPSS 26.0 (IBM, Armonk, NY). Data are presented as Mean ± SD unless 
noted otherwise. Statistical significance was set to p < 0.05. It was calculated that a minimum of 16 total 
participants in a crossover design (16 each group, BE and placebo) would enable 80% power to observe a 
3 to 5% change in FMD between the two groups10. An effect size analysis was performed using Cohen’s d 
and interpreted 0.2, 0.5, and 0.8 as small, medium, and large effect sizes, respectively11. 
 
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