Journal of Dietary Supplements

ISSN: 1939-0211 (Print) 1939-022X (Online) Journal homepage: https://www.tandfonline.com/loi/ijds20

Effects of Citrulline Malate and Beetroot Juice

Supplementation on Energy Metabolism and Blood
Flow During Submaximal Resistance Exercise

Eric T. Trexler, Dale S. Keith, Adam A. Lucero, Lee Stoner, Todd A. Schwartz,
Adam M. Persky, Eric D. Ryan & Abbie E. Smith-Ryan

To cite this article: Eric T. Trexler, Dale S. Keith, Adam A. Lucero, Lee Stoner, Todd A. Schwartz,
Adam M. Persky, Eric D. Ryan & Abbie E. Smith-Ryan (2019): Effects of Citrulline Malate and
Beetroot Juice Supplementation on Energy Metabolism and Blood Flow During Submaximal
Resistance Exercise, Journal of Dietary Supplements, DOI: 10.1080/19390211.2019.1650866

To link to this article: https://doi.org/10.1080/19390211.2019.1650866

Published online: 28 Aug 2019.

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JOURNAL OF DIETARY SUPPLEMENTS
https://doi.org/10.1080/19390211.2019.1650866

ARTICLE

Effects of Citrulline Malate and Beetroot Juice

Supplementation on Energy Metabolism and Blood
Flow During Submaximal Resistance Exercise
Eric T. Trexler, PhDa,b, Dale S. Keith, BAb, Adam A. Lucero, BScc, Lee Stoner, PhDa,b,
Todd A. Schwartz, DrPHd, Adam M. Persky, PhDe, Eric D. Ryan, PhDa,b, and
Abbie E. Smith-Ryan, PhDa,b
a
Human Movement Science Curriculum, Department of Allied Health Sciences, University of North
Carolina, Chapel Hill, NC, USA; bApplied Physiology Laboratory, Department of Exercise and Sport
Science, University of North Carolina, Chapel Hill, NC, USA; cSchool of Sport, Exercise and Nutrition,
Massey University, Wellington, NZ; dDepartment of Biostatistics, Gillings School of Global Public Health,
University of North Carolina, Chapel Hill, NC, USA; eEshelman School of Pharmacy, University of North
Carolina, Chapel Hill, NC, USA

ABSTRACT KEYWORDS
The ergogenic effects of citrulline malate (CitMal) and beetroot energy efficiency; exercise
juice (BEET) have been widely studied, but their effects on hyperemia; leg extension;
physiological outcomes related to resistance exercise are not fully nitric oxide; oxygen
consumption; vasodilation
understood. The purpose of this randomized, double-blind, cross-
over study was to investigate the effects of CitMal (8 g) and BEET
(400 mg nitrate) on blood pressure (BP), blood flow, and energy
efficiency during submaximal leg extension. Recreationally active
males (n ¼ 27; age: 22 ± 4 yrs) completed familiarization, followed
by three testing visits. Supine and standing BP were measured
upon arrival, followed by supplement ingestion, a 2-h rest period,
postsupplement BP measurement, and a bout of repeated sub-
maximal isotonic leg extensions at 25% of maximal voluntary
contraction torque. Diameter (aDIAM) and blood flow (aBF) of the
superficial femoral artery, and cross-sectional area (CSA) and echo
intensity (EI) of the vastus lateralis, were measured before and
after exercise via ultrasonography. Muscle blood flow (mBF) and
oxygen consumption (mVO2), along with whole-body energy
expenditure (EE) and respiratory exchange ratio (RER), were meas-
ured before and during exercise via indirect calorimetry and
near-infrared spectroscopy. Baseline RER values differed among
treatments (p ¼ 0.01); BEET was higher than CitMal (p ¼ 0.01) but
not PLA (p ¼ 0.58); CitMal and PLA were not significantly different
(p ¼ 0.12). No other measurements were significantly affected by
treatment (all p > 0.05). Results suggest that neither CitMal nor
BEET significantly influence resting BP, blood flow, or metabolic
efficiency during submaximal leg extension in recreationally
active males.

CONTACT Abbie E. Smith-Ryan, PhD abbsmith@email.unc.edu Department of Exercise and Sport Science,
University of North Carolina-Chapel Hill, 312 Woollen, CB #8700, Chapel Hill, NC 27599.
ß 2019 Taylor & Francis Group, LLC
2 E. T. TREXLER ET AL.

Introduction
Nitric oxide (NO) precursor supplements, such as arginine, citrulline, and beetroot juice
(BEET), are a popular class of dietary supplements that are typically marketed toward
physically active populations as ergogenic aids (Bloomer et al. 2010; 2010). A number of
recent studies have documented performance improvements following NO precursor
supplementation in a variety of exercise tasks, such as cycling (Bailey et al. 2009;
Lansley et al. 2011; Cermak et al. 2012), running (Lansley et al. 2011), rowing (Bond
et al. 2012), and resistance exercise (Perez-Guisado and Jakeman 2010; Wax et al. 2015;
Mosher et al. 2016; Wax et al. 2016). Nitric oxide is purported to elicit several physio-
logical responses that may influence resistance exercise performance, with evidence sug-
gesting that NO induces favorable changes in blood flow, exercise efficiency, and
muscle fatigue (Bailey et al. 2012). While NO precursor supplements are widely mar-
keted and used as ergogenic aids for resistance exercise, there is currently a paucity of
literature in this area, and gaps in the literature pertaining to effects of NO precursors
on physiological responses related to resistance exercise.
Nitric oxide is produced endogenously, but supplementation with NO precursors is
thought to increase the body’s capacity for NO production. Citrulline and BEET are
two of the most common NO precursor supplements, with studies generally using doses
of 3–6 g of citrulline (Trexler et al. 2019) and BEET products containing 5–9 mmol of
nitrate (Jones 2014). Vasodilation is the most widely known physiological effect of NO,
but supplementation with NO precursors has yielded mixed results on blood flow.
Nitric oxide induces relaxation of the vasculature by activating guanylyl cyclase, which
catalyzes the conversion of guanosine triphosphate (GTP) to cyclic guanosine mono-
phosphate (cGMP) (Guix et al. 2005). Exercise involving repetitive contractions of large
muscle groups increases metabolic demand of the active musculature, thereby increasing
the need for energy substrates and oxygen (Joyner and Casey 2015). Increased blood
flow to active musculature during exercise, known as exercise hyperemia, is therefore a
critical physiological process required to enable vigorous exercise, and NO precursors
may therefore influence exercise outcomes by modulating the magnitude or onset of
this hyperemic response. In rodents, five days of BEET supplementation has been
shown to augment the exercise-induced increase in blood flow to the active musculature
(Ferguson et al. 2013), but results of human studies have been comparatively less con-
clusive. Kim et al. (Kim et al. 2015) found no effect of a single dose of BEET supple-
mentation on brachial artery blood flow during graded handgrip exercise. By contrast,
Richards et al. (Richards et al. 2018) measured brachial artery blood flow during hand-
grip exercise with single-dose BEET supplementation and found a significant increase in
blood flow. One study investigating 14 days of L-citrulline ingestion (Gonzales et al.
2017) found an improvement in femoral artery blood flow during calf exercise in older
men, but not in older women, whereas another study (Kim et al. 2015) reported signifi-
cant increases in NO production without improvements in forearm blood flow follow-
ing single-dose citrulline supplementation.
Independent of its effects on blood flow, NO has also been shown to influence aer-
obic energy production and mitochondrial respiration (Larsen et al. 2011). Nitric oxide
and NO-derived compounds have direct effects on mitochondria and, at varying con-
centrations, have been shown to bind to cytochrome c oxidase (Brown and Cooper
 JOURNAL OF DIETARY SUPPLEMENTS 3

1994) and mitochondrial complex I (Clementi et al. 1998; Brown and Borutaite 2004)
and to enhance the efficiency of oxidative phosphorylation (Clerc et al. 2007). The half-
life of NO is less than a few seconds (Thomas et al. 2001); as such, attempts to exploit
the effects of NO on vasodilation and mitochondrial efficiency via dietary supplementa-
tion are restricted to precursors of NO, such as citrulline and dietary nitrate. Several
studies have investigated the effects of BEET and other sources of nitrate on energy effi-
ciency during aerobic exercise, as determined using various indices of oxygen consump-
tion (VO2) or oxygen uptake kinetics in response to exercise. Lansley et al. (Lansley
et al. 2011) found reductions in the oxygen cost of treadmill walking, moderate-intensity
running, and high-intensity running following four to six days of BEET supplementa-
tion. In two studies using sodium nitrate, improvements in energy efficiency during
cycling exercise were observed in young, healthy adults (Larsen et al. 2011) and young,
endurance-trained men (Larsen et al. 2007) following three days of supplementation.
While most studies evaluating nitrate-induced improvements in energy efficiency have
been carried out using aerobic exercise modalities, only one has used a leg extension task
similar to resistance exercise (Bailey et al. 2010). While an enhancement of energy efficiency
was observed after four to six days of supplementation, more research is required to replicate
this finding to determine whether energy efficiency enhancement may contribute to previ-
ously reported improvements in resistance exercise performance following six days of BEET
supplementation (Mosher et al. 2016). For citrulline-based supplements, effects on energy
efficiency during exercise are unclear. For example, Ashley et al. (Ashley et al. 2018) recently
found seven days of L-citrulline supplementation to favorably affect multiple indices of VO2
kinetics in men, but not in women. During a cycling time trial, seven days of L-citrulline
was not found to significantly affect VO2 response in comparison to a placebo treatment,
but the ratio of power output to oxygen consumption tended to be higher in the citrulline
condition (Suzuki et al. 2016). While both BEET (Mosher et al. 2016) and citrulline malate
(CitMal) (Perez-Guisado and Jakeman 2010; Wax et al. 2015; 2016) have been found to
delay fatigue during exhaustive resistance exercise, these supplements provide different NO
precursors, which target different NO production pathways that are stimulated and inhibited
by distinct physiological conditions (Bailey et al. 2012). More research is needed to deter-
mine whether these ingredients induce similar effects on energy efficiency, particularly with
respect to resistance exercise.
To fully understand the ergogenic potential of NO precursor supplements, more data
are needed to evaluate the effects of BEET and CitMal on energy efficiency and blood
flow during resistance. The purpose of the current study was to evaluate the effects of
CitMal and BEET supplementation on parameters of blood flow and metabolic effi-
ciency during submaximal leg extension exercise. It was hypothesized that CitMal and
BEET would both increase indices of blood flow and enhance energy efficiency in com-
parison to placebo (PLA).

Methods
Experimental design
The current study evaluated the effects of CitMal and BEET supplementation in a
randomized, double-blind, placebo-controlled, counterbalanced crossover design.
4 E. T. TREXLER ET AL.

Figure 1. Timeline of each testing visit. CitMal, citrulline malate; BEET, beetroot juice; PLA, placebo;
BP, blood pressure; FA, femoral artery; VL, vastus lateralis; NIRS, near-infrared spectroscopy.

Participants completed a familiarization visit, in which maximal voluntary leg extension

torque (MVC) was determined at a 90 knee angle. Participants completed three return
visits, separated by 5–10 days (mean ± standard deviation [SD]: 7.1 ± 0.9 days), to assess
the effects of supplementation on parameters of energy efficiency and blood flow during
submaximal leg extension exercise. The right leg was used for all measurements in all
visits. After 5 min of supine rest, blood pressure (BP) was measured with an automated
sphygmomanometer in supine and standing positions, then a treatment beverage
(CitMal, BEET, or placebo [PLA]) was consumed 2 h before exercise. Before the onset
of exercise, participants laid supine for 5 min, and resting measurements were obtained
for supine and standing BP, diameter (aDIAM) and blood flow (aBF) of the superficial
femoral artery, cross-sectional area (CSA) and echo intensity (EI) of the vastus lateralis
muscle (VL), muscle blood flow (mBF) and oxygen consumption (mVO2) of the VL,
and whole-body energy expenditure (EE) and respiratory exchange ratio (RER). Exercise
consisted of clusters of isotonic leg extensions, with 1 repetition every 4 s for approxi-
mately 8 min at an intensity of 25% MVC torque. The test visit timeline is presented in
Figure 1. Blood pressure, aBF, aDIAM, mBF, CSA, and EI were collectively measured to
assess the effects of supplementation on the control of blood flow and fluid accumula-
tion in the active musculature. Whole-body EE and mVO2 were measured to assess the
energy efficiency of exercise, and RER was measured to assess fuel utilization during
exercise. These assessments of blood flow, energy expenditure, and substrate utilization
were selected to evaluate several potential mechanisms by which acute NO precursor
supplementation has been purported to influence resistance exercise performance.

Participants
Healthy, recreationally active, nonsmoking male participants between the ages of 18 and 35
yrs participated in the current study. Participants were required to exercise (such as resist-
ance exercise, endurance exercise, or recreational sports) an average of at least 2 h per week
for at least 8 weeks preceding the study and to be free from diseases or injuries that would
contraindicate vigorous exercise or influence study outcomes. Participants could not have
 JOURNAL OF DIETARY SUPPLEMENTS 5

Table 1. Participant characteristics (n ¼ 27) and dietary intake information (n ¼ 25 logs returned).
Variable Mean SD Min Max
Age (yr) 22 4 18 35
Height (cm) 178.4 6.8 166.3 189.0
Weight (kg) 78.9 12.5 62.5 104.2
MVC (Nm) 222 43 150 331
VL FT (cm) 0.5 0.2 0.3 1.1
VL MT (cm) 2.8 0.3 2.3 3.5
KCald-1 2,558.0 698.7 1,173.0 4,723.0
CHO (gd-1) 279.9 90.0 107.7 526.5
FAT (gd-1) 101.3 33.8 48.8 154.5
PRO (gd-1) 131.1 42.0 70.5 262.5
Nitrate (mgd-1) 115.1 98.1 36.1 410.9
SD, standard deviation; Min, minimum value; Max, maximum value; MVC, maximum voluntary contraction torque; VL,
vastus lateralis; FT, subcutaneous fat thickness at site of probe placement; MT, muscle thickness at site of probe place-
ment; Kcal, kilocalories; CHO, carbohydrate; PRO, protein.

gained or lost  4.5 kg within 8 weeks of enrollment or regularly taken prescription medica-
tions or dietary supplements that would influence study outcomes within 8 weeks of enroll-
ment. Participants were excluded if they had participated in a clinical trial involving
substantial diet or exercise modifications in the previous 8 weeks, consumed more than
three alcoholic drinks per day, or used recreational drugs within the past month. To obtain
clear muscle blood flow data, participants were also required to have a subcutaneous adi-
pose tissue thickness of < 12.0 mm at the site of sensor placement.
All testing visits occurred at the same time of day (±1 h), following at least 4 h of fasting.
Participants were instructed to refrain from strenuous exercise and alcohol consumption
within 24 h of testing visits and from caffeine consumption within 12 h. Participants were
instructed not to smoke throughout the study, to refrain from using antibacterial mouth-
wash the day of testing, and to abstain from chewing gum and brushing their teeth within at
least 8 h of visits (van Maanen et al. 1996). Throughout the study, participants were encour-
aged to maintain their normal dietary and physical activity habits, and typical dietary intakes
were evaluated via completion of a single three-day food log. Diet logs were analyzed for
macronutrient intake using The Food Processor software (ESHA Research, OR, USA) and
daily nitrate intake using food nitrate content estimates as previously described (Jonvik et al.
2017). All participants provided written informed consent prior to participation, and all
study procedures were approved by the Biomedical Institutional Review Board of the
University of North Carolina at Chapel Hill.
Thirty subjects enrolled in the current study. Three withdrew prior to the first testing
visit; one withdrew after the first testing visit was completed, but prior to the second.
Participants providing reasons for withdrawal cited either schedule constraints or inju-
ries unrelated to the study; attrition did not appear to be related to any specific distin-
guishing characteristics. In total, 27 participants completed at least one testing visit;
data missingness was minimal, with all omnibus tests including at least 76 of 79 possible
observations. Demographic characteristics of the sample are presented in Table 1.

Familiarization visit
Prior to testing visits, participants completed a familiarization visit that began by
obtaining written informed consent. Participants then completed a health history
6 E. T. TREXLER ET AL.

questionnaire and received instructions for pre-visit guidelines. Participants were then
positioned on an isokinetic dynamometer (HUMAC NORM, Computer Sports
Medicine Inc., MA, USA). The backrest was reclined to provide a hip angle of 110 to
allow for unimpeded blood flow to the leg. Dynamometer settings were recorded and
replicated at all subsequent visits to ensure consistent placement and positioning. The
lever arm of the dynamometer was locked in place with the right knee placed in 90 of
flexion, and participants practiced three escalating submaximal isometric contractions of
50%, 75%, and 90% estimated effort to warm up. Participants then completed three iso-
metric maximal voluntary contractions (MVCs) for 3–5 s, with 1 min of rest between
attempts. The highest of the three attempts was used to calculate the resistance for the
submaximal exercise protocol (25% of highest MVC). Participants then completed leg
extension familiarization, which consisted of three sets of isokinetic leg extension. In
each set, participants completed 30 concentric leg extensions from a 90 knee angle to
full leg extension at 180 s1, with passive leg flexion at 90 s1, and 1 min of rest
between sets. Throughout the study, all leg extension tasks were completed with the
arms folded across the chest and a seatbelt tightly fastened across the chest and waist.
Participants returned 2–10 days following familiarization (mean ± SD: 6.3 ± 2.0 days) for
the first testing visit.

Supplementation
Treatment sequence was randomly assigned in a counterbalanced manner using
Random Allocation Software (Isfahan, Iran). Randomized treatment sequences included
all permutations for the three treatment arms: (1) 70-mL beetroot juice beverage yield-
ing 400 mg dietary nitrate (BEET; Beet It Sport, James White Drinks Ltd., Ipswich, UK),
(2) placebo (PLA), and (3) 8 g of unflavored citrulline malate (CitMal; 2:1 ratio,
BulkSupplements.com, NV, USA) mixed into the placebo beverage. As previously
described (Mosher et al. 2016), blackcurrant juice (70 mL; Ribena, Lucozade Ribena
Suntory Ltd., Uxbridge, UK) was used as the placebo beverage; it is similar to BEET in
taste, appearance, and macronutrient content but has negligible nitrate content.
Additional sweetener (Crystal Light; Kraft Foods, IL, USA) and lemon juice were
included to further mask flavors. To maintain a double-blinded design, treatments were
mixed in opaque containers by an individual that was not present for supplement inges-
tion or testing. Treatments were consumed in the laboratory 2 h prior to exercise testing
as peak blood levels of NO precursors are achieved approximately 1.4–2.3 h after citrul-
line ingestion (Schwedhelm et al. 2008) and 2–3 h after BEET ingestion (Wylie
et al. 2013).

Height, weight, and blood pressure

Upon arrival to the laboratory for testing visits, height and weight were measured in
light athletic clothing, without shoes, using a stadiometer (Perspective Enterprises PE-
AIM-101, MI, USA) and calibrated electronic scale (Health-O-Meter 2101KL, IL, USA).
Participants then rested quietly for 5 min in a supine position, and supine BP was meas-
ured using an automated sphygmomanometer (Omron HEM-711DLX, IL, USA) on the
 JOURNAL OF DIETARY SUPPLEMENTS 7

left arm. Participants then assumed a standing position, and standing BP was measured
with the arm fully relaxed and supported at chest level. The supplement was provided
following BP measurement.

Ultrasound: Superficial femoral artery blood flow, vastus lateralis imaging

Doppler ultrasonography (Logiq-e, GE Healthcare, IL, USA) was used to assess vessel
diameter and blood flow through the superficial branch of the femoral artery of the
right leg. The probe (12LRS, 5–13 mhz) was positioned 1–3 cm distal to the location
where the femoral artery bifurcates into the superficial and deep branches. For each
image, a minimum of four cardiac cycles were recorded in duplex mode, with real-time
imaging of the vessel and the spectral waveform velocity profile. Artery diameter
(aDIAM) and arterial blood flow (aBF) were estimated using the measure function in
the device’s default software, as previously reported (Roelofs et al. 2017). Velocity values
were averaged between up to four consecutive cardiac cycles for each image. At least
two images were obtained at each timepoint, with aDIAM and aBF values from both
images averaged for analysis. Test-retest reliability using these methods for brachial
artery diameter (intraclass correlation coefficient [ICC] ¼ 0.82, standard error of meas-
urement [SEM] ¼ 0.03 cm) and blood flow (ICC ¼ 0.86, SEM ¼ 5.92 mLmin1) has been
reported previously for this lab (Roelofs et al. 2017).
Brightness-mode ultrasound was also used to assess changes in VL CSA and EI due
to exercise hyperemia. A panoramic scan was performed at the midpoint of the VL, and
images of the VL were traced and analyzed offline by the same trained technician using
ImageJ software (National Institute of Health, MD, USA, Version 1.37). Each image was
outlined using the polygon tool to capture as much of the muscle belly as possible with-
out capturing the epimysium. Muscle CSA was estimated as the area of this polygon;
echo intensity was calculated as the average pixel brightness within this polygon, rang-
ing from 0 (darkest) to 255 (brightest). Echo intensity values were corrected for sub-
cutaneous fat thickness as described by Young et al. (Young et al. 2015), with fat
thickness represented as the average of three subcutaneous fat thickness measurements
taken at the medial, middle, and lateral aspects of the VL. At least two images were cap-
tured at each timepoint, with values averaged for analysis. Test-retest reliability for VL
CSA (ICC ¼ 0.87, SEM ¼ 2.12 cm2) and EI (ICC ¼ 0.74, SEM ¼ 4.58 arbitrary units
[AU]) have been previously reported for this lab using this methodology (Melvin et al.
2014). Resting scans were performed following at least 5 min of supine rest. Postexercise
scans were also obtained in the supine position, with vessel scans occurring 5 min fol-
lowing the cessation of exercise (Martin et al. 2017).

Near-infrared spectroscopy: Muscle blood flow and oxygen consumption

To assess muscle blood flow (mBF) and muscle oxygen consumption (mVO2) measures,
a continuous wave near-infrared spectroscopy (NIRS) device was used (PortaLite,
Artinis Medical Systems BV, The Netherlands). This device uses near-infrared light to
detect relative changes in oxygenated (O2Hb), deoxygenated (HHb), and total Hb
(t[Hb] ¼ [O2Hb þ HHb]) in the VL. The NIRS probe was fixed to the skin two-thirds
8 E. T. TREXLER ET AL.

from the top of the right VL, parallel to the muscle fibers and in the center of the
muscle belly. The probe was covered with a custom protective shield and tape to block
light and allow the probe to move with the skin during contractions. To estimate mBF
and mVO2, an inflatable blood pressure cuff was placed around the proximal thigh
upstream of the NIRS probe. The cuff was rapidly inflated to occlude venous flow to
allow mBF estimation, or both arterial and venous flow to allow mVO2 estimation.
For venous occlusions, the cuff was inflated to a subsystolic pressure (60 mmHg) to
occlude venous return without obstructing arterial flow. As such, venous volume
increases in direct proportion to arterial flow, allowing measurement of mBF by calcu-
lating the slope of the change in t[Hb]. This slope was converted to units of mLmin1
per 100 mL of blood using the following equation (Lucero et al. 2017):
2 3
Dt ½Hb  60
mBF ðmL  min1
 100 mL 1 Þ
¼4
  10005=10
½Hb1000
4

To obtain a value for blood hemoglobin concentration [Hb], a venous blood sample
was obtained at rest, and a droplet was analyzed using an automated hemoglobin
analyzer (HemoPoint H2, Stanbio Laboratory, TX, USA). In one instance, a resting
blood sample could not be obtained; the average [Hb] from the participant’s other visits
was imputed for NIRS analysis.
To assess mVO2, the cuff was inflated to a suprasystolic pressure (280 mmHg)
to simultaneously occlude venous and arterial flow. This results in an increase of [HHb]
and decrease in [O2Hb]; the blood volume-corrected slope of [HHb] was converted to
mLO2min1 per 100 grams of tissue using the following equation (Lucero et al. 2017):
 
 1 1
 D½HHb  60
mVO2 mLO2  min  100 g ¼ abs  4  22:4=1000
10  1:04

Resting measures of mBF and mVO2 were taken with participants seated on the
dynamometer with the leg fixed in a neutral position (150 knee angle) and the VL fully
relaxed. Four 15-s venous occlusions were applied for resting mBF measurements, with
45 s of rest between measurements. After resting mBF measurements, resting mVO2 was
measured twice, using 15–30 s suprasystolic occlusions with 1–2 min of rest between
measurements. Occlusion pressures were determined on the basis of observations
obtained during pilot testing and data collection for a previous study by the current
research group using the same protocol (Lucero et al. 2017), using NIRS and Doppler
ultrasound to assess venous and arterial flow at various occlusion pressures. Heart
rhythm was continuously recorded via electrocardiography, with slope values obtained
from a single cardiac cycle of each venous occlusion (Cross and Sabapathy 2017). All
NIRS data were collected at a sampling frequency of 50 Hz with a differential pathlength
factor of 4.0 Analysis was done using transmitter 2 with a theoretical penetration
distance of 2.0 cm. The reliability of the described method for mBF (ICC ¼ 0.90) and
mVO2 (ICC ¼ 0.96) using leg extensions at 25% of MVC torque has been previously
confirmed for this lab (Lucero et al. 2017).
 JOURNAL OF DIETARY SUPPLEMENTS 9

Indirect calorimetry
To assess energy expenditure (EE; KCal) and RER (AU), respiratory gases were
collected continuously for 15 min of rest and throughout the submaximal leg exten-
sion test (PRE). Participants were seated on the dynamometer and connected to an
indirect calorimeter (TrueOne 2400, ParvoMedics, Inc., UT, USA) by a mouthpiece
and hose. For resting values, the first 5 min of data were discarded, with the final
10 min of rest used for analysis. Exercise values were obtained from the 8-min period
in which leg extension occurred. Expired gas values were averaged every 15 s and used
to calculate EE and RER by the device’s default software, and the rate of EE
(KCald1) was converted to KCal burned in 8 min by dividing by 180. Test–retest
reliability values using similar methodology for resting EE (ICC ¼ 0.94,
SEM ¼ 125.6 kcald1) and RER (ICC ¼ 0.83, SEM ¼ 0.03 AU) have previously been
reported for this lab (Hirsch et al. 2016).

Exercise protocol
Following resting measurements, participants completed the submaximal leg extension
protocol. The dynamometer was set to isotonic mode, with resistance set at 25% of
MVC torque (Lucero et al. 2017). Participants completed one concentric repetition
every 4 s, extending the leg from a 90 knee angle to a 150 knee angle, then relaxing as
the leg passively returned to the flexed position (90 ). Participants completed 3 min
of repeated contractions to achieve a steady state. Immediately following the final
repetition of this 3-min bout, the limb was supported at a 150 knee angle with the leg
musculature relaxed, and a 10-s venous occlusion was used to assess mBF. Exercise was
resumed for 45 s to reestablish a steady state, with participants alternating between
45-s clusters of contractions and venous occlusions until four mBF measurements
were obtained.
Following the fourth mBF measurement, the occlusion pressure was increased to
a suprasystolic pressure (280 mmHg) to allow for mVO2 measurement, and two more
clusters of contractions (45 s) and inflations occurred. In total, the protocol consisted of
six resting measurements (4 mBF, 2 mVO2) and six exercise measurements (4 mBF,
2 mVO2). Multiple measurements were taken for each outcome to allow values to be
averaged after removal of any unclear signals. Immediately following the final
NIRS measurement, the participant was laid supine on a nearby scanning table for
postexercise ultrasound measurements.

Statistical analysis
To verify that the exercise protocol elicited increases in blood flow and energy expend-
iture within each period, paired sample t tests were conducted separately to compare
resting and exercise values for each outcome of interest using PROC TTEST (SAS
Software, NC, USA). To evaluate the effect of supplementation, data were analyzed
using a series of general linear mixed models. Random intercept models were fitted,
with subject identified as a random effect. Preliminary models were fitted separately to
confirm nonsignificance of carryover, sequence, period  treatment, and habitual
10 E. T. TREXLER ET AL.

Table 2. Resting blood pressure measurements after supplementation.

Mean (mmHg) SE F (treatment) p (treatment)
Supine SBP F(2, 48) ¼ 0.30 0.74
CitMal 117.3 1.2
PLA 118.5 1.1
BEET 117.6 1.2
Supine DBP F(2, 49.6) ¼ 0.74 0.48
CitMal 68.4 1.1
PLA 70.0 1.1
BEET 69.9 1.1
Standing SBP F(2, 44.9) ¼ 0.38 0.68
CitMal 110.3 1.8
PLA 110.6 1.8
BEET 108.7 1.8
Standing DBP F(2, 47.9) ¼ 0.17 0.84
CitMal 66.4 1.0
PLA 66.7 1.0
BEET 67.1 1.0
SBP, systolic blood pressure; DBP, diastolic blood pressure; Mean, adjusted least squares mean; SE, standard error.
Data presented as least square mean ± SE. A significant carryover effect was observed for standing DBP (p < 0.001),
but analysis stratified by previous treatment revealed no significant treatment effect in the period following the
provision of CitMal, PLA, or BEET (all p > 0.05). Similarly, analysis of data from period 1 only revealed no significant
treatment effect (p ¼ 0.42).

nitrate  treatment interaction effects. Separate reduced models were then fitted to
assess the effects of treatment on each outcome variable, with fixed effects including
period, treatment, and the baseline value of the dependent variable for the correspond-
ing period as a covariate. Model residuals were visually assessed to confirm normality
and homoscedasticity. Resting blood pressure variables were compared using presupple-
mentation blood pressure values as a covariate. For all other variables, outcomes
measured during or immediately following the submaximal exercise bout were com-
pared using resting values as a covariate. Data missing due to technician or equipment
error were assumed to be missing completely at random, with all omnibus tests
including at least 76 of 79 possible observations. One observation was missing for NIRS
outcomes and arterial ultrasound measurements; two observations were missing from
resting indirect calorimetry outcomes and three for indirect calorimetry during exercise.
In the event of a significant overall treatment effect, pairwise comparisons were adjusted
for multiplicity using the Tukey-Kramer method. As a secondary analysis, baseline
values related to blood flow and indirect calorimetry variables were compared to
evaluate treatment effects on resting values. Statistical analyses were conducted using
SAS PROC MIXED, with the a priori criterion for significance set at a ¼ 0.05.
Descriptive demographic data are presented as mean ± standard deviation, as well
as minimum and maximum; findings from the general linear mixed models are
presented as adjusted least squares mean ± standard error.

Results
Blood pressure
Treatment did not significantly affect BP in the supine or standing posture (all p > 0.05;
Table 2).
 JOURNAL OF DIETARY SUPPLEMENTS 11

Exercise responses
Compared to resting values, exercise significantly increased CSA, aBF, mBF, mVO2, EE,
and RER within each period (all p < 0.05). Exercise induced increases in EI within each
period (D ¼ 0.88–1.73 AU), to varying degrees of statistical significance (p ¼ 0.01–0.23).
Significant changes within each period were not observed for aDIAM (all p > 0.05).

Ultrasonography
Significant differences among treatments were not observed for CSA (F[2, 48] ¼ 1.08,
p ¼ 0.35; CitMal: 31.7 ± 0.2, PLA: 32.1 ± 0.2, BEET: 32.0 ± 0.2 cm2). Similarly, no treat-
ment effect was observed for fat-corrected muscle EI (F[2, 48.6] ¼ 1.93, p ¼ 0.16;
CitMal: 91.4 ± 0.66, PLA: 91.9 ± 0.65, BEET: 93.2 ± 0.66 AU). Baseline resting values did
not differ among treatments for aDIAM (F[2, 47.3] ¼ 0.30, p ¼ 0.74) or aBF (F[2,
47.8] ¼ 0.63, p ¼ 0.53). For submaximal exercise responses, the effect of treatment on
aDIAM was not significant (F[2, 43.4] ¼ 0.88, p ¼ 0.42; CitMal: 0.68 ± 0.01, PLA:
0.68 ± 0.01, BEET: 0.70 ± 0.01 cm). A significant carryover effect was observed (p ¼ 0.05),
so data were stratified by previous treatment for exploratory between-group (not
within-person) analysis. No significant treatment effect was observed in the periods fol-
lowing CitMal or PLA provision (both p > 0.05), but aDIAM was greater in the BEET
condition compared to CitMal in the period following PLA ingestion (0.71 ± 0.01 versus
0.67 ± 0.01 cm, p ¼ 0.02). When analyzed using data only from period 1 in between-
group fashion, the treatment effect was not significant (p ¼ 0.055), but least squares
means followed a similar trend, with higher values for BEET (0.69 ± 0.01 cm) in com-
parison to CitMal (0.66 ± 0.01 cm). For submaximal exercise responses, artery flow was
not significantly affected by treatment (F[2, 48.3] ¼ 0.75, p ¼ 0.48; CitMal: 169.0 ± 9.6,
PLA: 158.7 ± 9.6, BEET: 156.9 ± 9.6 mLmin1).

Near-infrared spectroscopy
Baseline resting values did not differ among treatments for mBF (F[2, 47.4] ¼ 0.14,
p ¼ 0.87) or mVO2 (F[2, 47.9] ¼ 0.65, p ¼ 0.53). For submaximal exercise responses, a
significant treatment effect was not observed for mBF (F[2, 45.5] ¼ 0.11, p ¼ 0.90;
CitMal: 3.78 ± 0.26, PLA: 3.72 ± 0.26, BEET: 3.77 ± 0.26 mLmin1100 ml1). Similarly,
submaximal exercise mVO2 was not significantly affected by treatment (F[2,
46.2] ¼ 0.85, p ¼ 0.44; CitMal: 1.15 ± 0.11, PLA: 1.16 ± 0.11, BEET: 1.19 ± 0.11
mLO2min1100g1). Near-infrared spectroscopy values during submaximal exercise
are presented in Figure 2.

Indirect calorimetry
Baseline resting EE values did not differ among treatments (F[2, 47.1] ¼ 1.40, p ¼ 0.26).
The effect of treatment on exercise EE was not significant (F[2, 47.4] ¼ 2.10, p ¼ 0.13;
CitMal: 21.6 ± 0.5, PLA: 20.6 ± 0.5, BEET: 21.6 ± 0.5 kcal). Baseline RER values differed
among treatments (F[2, 47.9] ¼ 4.68, p ¼ 0.01); BEET was higher than CitMal
(0.78 ± 0.01 versus 0.75 ± 0.01 AU; p ¼ 0.01) but not PLA (0.77 ± 0.01 AU, p ¼ 0.58).
12 E. T. TREXLER ET AL.

Figure 2. Effects of treatment on muscle blood flow (mBF) and oxygen consumption (mVO2) during
exercise, as measured via near-infrared spectroscopy. Data are presented as adjusted least squares
mean ± standard error.

CitMal and PLA were not significantly different (p ¼ 0.12). Treatment did not
significantly affect exercise RER (F[2, 47.1] ¼ 0.45, p ¼ 0.64; CitMal: 0.80 ± 0.01, PLA:
0.79 ± 0.01, BEET: 0.79 ± 0.01 AU). Indirect calorimetry values during submaximal
exercise are presented in Figure 3.

Discussion
Numerous studies have reported ergogenic effects on exercise tasks, including time to
exhaustion, time-trial performance, and repetitions to fatigue from NO precursor
supplements such as CitMal and BEET (Bailey et al. 2009; Perez-Guisado and Jakeman
2010; Lansley et al. 2011; 2011; Bond et al. 2012; Cermak et al. 2012; Wax et al. 2015;
Mosher et al. 2016; Wax et al. 2016). Ergogenic effects are thought to be mediated by
NO, which is purported to enhance blood flow and energy efficiency during exercise.
However, there is a lack of research specifically linking these mechanisms to resistance-
type exercise, and more data are needed to elucidate the mechanisms underlying
previously reported improvements in resistance exercise performance following NO
precursor supplementation. The current findings do not suggest that acute ingestion
 JOURNAL OF DIETARY SUPPLEMENTS 13

Figure 3. Effects of treatment on whole-body energy expenditure (EE) and respiratory exchange
ratio (RER) during exercise, as measured via indirect calorimetry. Kcal, kilocalories; AU, arbitrary units.
Data are presented as adjusted least squares mean ± standard error.

of CitMal or BEET prior to exercise significantly alter blood flow or energy efficiency
during submaximal leg extension in recreationally active males.
Parameters pertaining to blood pressure were not affected by CitMal or BEET in the
current study. Neither systolic nor diastolic BP was influenced by treatment; while both
CitMal and BEET have been shown to reduce blood pressure, effects are less likely to
be observed in young, active, normotensive participants (Kim et al. 2015; Wax et al.
2015), such as the current sample. Notably, NO synthesis and endothelial function often
decrease with age (Toprakci et al. 2000; Seals et al. 2011), which may explain
age-dependent effects of NO precursor supplementation. Similarly, supplement-induced
alterations of BP are more likely to be observed in individuals with elevated BP at
baseline, with underlying disruptions in NO metabolism and/or endothelial function.
Such disruptions may impair the regulation of blood pressure and exercise hyperemia
in older and hypertensive individuals, whereas young, normotensive individuals are
more likely to have sufficient regulation of blood pressure and exercise hyperemia. As
such, NO precursor supplements may be particularly efficacious for blood pressure and
blood flow outcomes in older and hypertensive individuals. For example, single-dose
BEET supplementation was recently shown to reduce systolic (7.9 mmHg) and
diastolic (5.7 mmHg) BP after acute ingestion, but the mean age of the sample was
14 E. T. TREXLER ET AL.

64 years, and the average BP was 133/88.6 mmHg at baseline (Raubenheimer et al.
2017). Blood pressure results of the current study are favorable for normotensive
individuals interested in supplementing with NO precursors, as neither BEET nor CitMal
induced resting hypotension or postural hypotension in this normotensive sample.
Similarly, aDIAM, aBF, and mBF were not influenced by CitMal or BEET. While
Richards et al. (Richards et al. 2018) observed approximately 12%–13% increases in
brachial artery blood flow during handgrip exercise following single-dose BEET ingestion,
the current findings are supported by studies in which both citrulline (Churchward-Venne
et al. 2014) and BEET (Kim et al. 2015; Craig et al. 2018) have failed to enhance conduit
artery blood flow during or immediately following exercise. Although Richards et al.
(Richards et al. 2018) evaluated a sample of young, healthy participants, the reported
increase in blood flow was observed after giving a nitrate dosage roughly two to three times
higher than that in the current study. Lack of significant blood flow effects in the current
study may also be related, to some extent, to the use of a young, healthy sample as single-
dose BEET supplementation has been shown to increase the compensatory vasodilator
response to hypoxic handgrip exercise in older adults (age: 64 ± 2 yr) but not in younger
adults (age: 25 ± 1 yr) (Casey et al. 2015). While aDIAM was not affected by supplementa-
tion, a carryover effect was observed; follow-up testing revealed that aDIAM was higher in
BEET than CitMal in the period following PLA ingestion. While this finding warrants fur-
ther investigation, it should be noted that stratifying analysis by previous treatment is
exploratory and drastically reduces the sample size of each statistical test, which increases
the likelihood of a statistically significant finding being spurious (Button et al. 2013).
With regard to mBF, the current findings are supported by previous research demon-
strating no effect of single-dose citrulline supplementation on microvascular circulation, as
measured via contrast-enhanced ultrasound (Churchward-Venne et al. 2014). Other stud-
ies have reported significant effects of one to three days of BEET supplementation on the
microvasculature as measured via NIRS, in the absence of venous or arterial occlusions
(Aucouturier et al. 2015; de Oliveira et al. 2017); both used higher nitrate doses than the
current study, and methodological differences in assessment of microvascular function
present challenges for making between-study comparisons. A strength of the current study
is the use of intermittent venous and arterial occlusions in conjunction with NIRS, which
enhances the ability to extrapolate localized hemoglobin fluctuations with indices of mBF
and mVO2 for inferences regarding microvascular circulation and energy efficiency.
Previous research has documented acute increases in muscle CSA and EI following acute
resistance exercise (Hill et al. 2018), reflecting a pooling of intramuscular fluid due to exer-
cise hyperemia. However, the current study found no effect of CitMal or BEET on these
indices of muscular fluid accumulation. These findings mirror the results of Gonzalez et al.
(Gonzalez et al. 2018), who found that single-dose CitMal supplementation had no effect
on the postexercise increase in muscle thickness that was observed. In summary, null
effects on all blood flow parameters in the current study may be attributable to the use of a
young, healthy, active sample in which endothelial function is unlikely to be impaired
(Casey et al. 2015; Kim et al. 2015). These results do not support the hypothesis that altera-
tions in blood flow are a primary mechanism explaining previous findings in which acute
NO precursor supplementation has enhanced resistance exercise performance in young,
healthy individuals (Perez-Guisado and Jakeman 2010; Wax et al. 2015; 2016).
 JOURNAL OF DIETARY SUPPLEMENTS 15

Indices of energy expenditure and oxygen consumption, measured via indirect calor-
imetry and NIRS, were not influenced by treatment in the current study. These findings
suggest that supplementation did not significantly influence energy efficiency during
exercise, which is a notable ergogenic mechanism referenced in the NO precursor litera-
ture pertaining to endurance exercise. A modest elevation in resting RER was observed
following BEET consumption in comparison to CitMal, which indicates a shift toward
increased reliance on glucose metabolism with BEET and a shift toward fat metabolism
with CitMal. A similar RER elevation following three days of nitrate supplementation
was observed during exercise by Larsen et al. (Larsen et al. 2011), who noted that
a change of this magnitude was of minimal physiological significance. With regard to
CitMal, Bendahan et al. (Bendahan et al. 2002) documented an increase in the relative
contribution of aerobic metabolism following multiple days of supplementation, which
may result in a modest RER reduction as observed in the current study. Furthermore,
these effects on RER were observed only at rest in the current study, were not signifi-
cantly different from PLA, and were not accompanied by changes in total EE, which
limit the practical significance of the findings. While multiday (three to six days) supple-
mentation interventions with BEET have been shown to enhance the energetic efficiency
of exercise using a variety of exercise modalities and measurement methodologies
(Larsen et al. 2007; Bailey et al. 2010; Lansley et al. 2011), the mechanism underlying
these observations is not fully understood. Larsen et al. (Larsen et al. 2011) have
proposed that increased exercise efficiency is related to increased coupling of mito-
chondrial respiration, as evidenced by improved oxidative phosphorylation efficiency
and decreased state 4 respiration. Such changes in mitochondrial efficiency are likely
related to downregulation of uncoupling protein 3 (UCP-3) and adenosine nucleotide
translator (ANT); alterations in protein structure or expression are more likely to be
affected by multiple-day dosing strategies in comparison to a single dose (Larsen et al.
2011), which may explain the null findings in the current study. However, Whitfield
et al. (Whitfield et al. 2016) recently found seven days of BEET supplementation
to reduce whole-body VO2 without altering indices of mitochondrial efficiency, which
suggests that an alternative mechanism may explain previously reported improvements
in exercise efficiency. In summary, the current results do not support the hypothesis
that alterations of energy efficiency or substrate utilization explain previously reported
improvements in repetitions to fatigue following acute NO precursor supplementation
(Perez-Guisado and Jakeman 2010; Wax et al. 2015, 2016).
It has been proposed that exercise efficiency improvements may relate to alterations
in muscle calcium kinetics, thereby reducing the ATP cost of muscle contraction (Bailey
et al. 2010; Hernandez et al. 2012). Acute nitrate supplementation, particularly with doses
of 8.8 mmol and above, may enhance muscle function by increasing sarcoplasmic reticulum
calcium release (via nitrosylation of ryanodine receptors) and/or by increasing myofibrillar
calcium sensitivity (via activation of guanylyl cyclase and subsequent elevation of cGMP)
(Coggan and Peterson 2018). Chronic supplementation may confer additional effects; after
seven days of nitrate supplementation, Hernandez et al. (Hernandez et al. 2012) observed
upregulation of calcium-handling proteins in muscle (calsequestrin 1 and dihydropyridine
receptor), whereas other research has shown increased force production in the absence of
changes in these proteins following seven days of supplementation (Whitfield et al. 2017).
16 E. T. TREXLER ET AL.

While more research is needed to determine the exact mechanisms by which NO may
modulate exercise efficiency, these effects appear to be more reliably observed using higher
and more prolonged dosing strategies than in the current study.
The primary limitation of the current study is the dosing protocol, which consisted of a sin-
gle dose of CitMal (8 g) or BEET (400 mg nitrate) prior to exercise. This approach allows for
observation of the acute effects of a single ingestion of citrulline and nitrate doses that are likely
to be used in products marketed toward general and athletic populations. However, as
reviewed by Jones (Jones 2014), the BEET literature suggests that more pronounced findings
may be observed with higher BEET dosages or more chronic, daily supplementation periods.
Habitual dietary intake was observed using a single three-day diet log; while participants were
instructed to maintain their typical dietary habits, it is possible that such habits could have fluc-
tuated throughout the study. In addition, preliminary statistical tests suggested that carryover
effects were present for standing DBP and aDIAM, despite 5–10 days of washout between test-
ing visits. Orally ingested CitMal or BEET would be readily cleared from systemic circulation
within this timeframe, but further research may be warranted to determine the time course in
which CitMal or BEET may modestly influence the function of the vasculature following sup-
plementation. Additional research is needed to build upon the current findings by incorporat-
ing other key assessments related to resistance exercise performance, such as perceived
exertion and indices of maximal contractile properties of muscle.

Conclusions
The current data do not support the hypothesis that a single dose of CitMal (8 g) or
BEET (400 mg nitrate) enhances blood flow or energy efficiency during submaximal leg
extension in young, recreationally active males. Previous studies have reported improve-
ments in resistance exercise performance, measured as repetitions to fatigue, following
acute NO precursor supplementation. Authors have speculated that such ergogenic
effects may be attributable to alterations in blood flow, energy efficiency, or substrate
utilization, but the current results do not support the hypothesis that these parameters
are significantly altered by acute CitMal or BEET supplementation. Acute effects of NO
precursor supplements on muscle contractile function, potentially mediated by altera-
tions in sarcoplasmic reticulum calcium kinetics and myofibrillar calcium sensitivity,
may warrant further research. As research demonstrating ergogenic effects of NO pre-
cursor supplements on resistance exercise performance continues to emerge (Perez-
Guisado and Jakeman 2010; Wax et al. 2015; Mosher et al. 2016; Wax et al. 2016),
more focused research is needed to elucidate the mechanisms underlying these effects.

Acknowledgments
The authors thank Shawn Ahuja and Casey Greenwalt for their assistance and all of the research
participants for their time and effort.

Declaration of interest
The authors declare no conflicts of interest. The authors alone are responsible for the content
and writing of the article.
 JOURNAL OF DIETARY SUPPLEMENTS 17

Funding
The current study was funded by the National Strength and Conditioning
Association Foundation.

Notes on contributors
Eric T. Trexler, PhD, is a research assistant in the Applied Physiology Laboratory at the
University of North Carolina at Chapel Hill.
Dale S. Keith, BA, is a research assistant in the Applied Physiology Laboratory at the University
of North Carolina at Chapel Hill.
Adam A. Lucero, BSc, is a doctoral candidate in the School of Sport, Exercise and Nutrition at
Massey University.
Lee Stoner, PhD, is an assistant professor of exercise physiology at the University of North
Carolina at Chapel Hill.
Todd A. Schwartz, PhD, is a research associate professor of biostatistics at the University of
North Carolina at Chapel Hill.
Adam M. Persky, PhD, is a clinical professor of pharmacy at the University of North Carolina at
Chapel Hill.
Eric D. Ryan, PhD, is an associate professor of exercise physiology at the University of North
Carolina at Chapel Hill.
Abbie E. Smith-Ryan, PhD, is an associate professor of exercise physiology and director of the
Applied Physiology Laboratory at the University of North Carolina at Chapel Hill.

ORCID
Abbie E. Smith-Ryan http://orcid.org/0000-0002-5405-304X

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