Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41
DOI 10.1186/s12970-015-0102-y

RESEARCH ARTICLE

Open Access

Effects of powdered Montmorency tart
cherry supplementation on an acute boutof intense lower body strength exercise inresistance trained males
Kyle Levers1, Ryan Dalton1, Elfego Galvan1, Chelsea Goodenough1, Abigail O’Connor1, Sunday Simbo1,
Nicholas Barringer2, Susanne U. Mertens-Talcott3, Christopher Rasmussen1, Mike Greenwood1, Steven Riechman4,
Stephen Crouse5 and Richard B. Kreider1*
Abstract
Background: The purpose of this study was to examine whether short-term ingestion of a powdered tart cherry
supplement prior to and following intense resistance-exercise attenuates muscle soreness and recovery strength
loss, while reducing markers of muscle damage, inflammation, and oxidative stress.
Methods: Twenty-three healthy, resistance-trained men (20.9 ± 2.6 yr, 14.2 ± 5.4 % body fat, 63.9 ± 8.6 kg FFM) werematched based on relative maximal back squat strength, age, body weight, and fat free mass. Subjects were randomly
assigned to ingest, in a double blind manner, capsules containing a placebo (P, n = 12) or powdered tart cherries[CherryPURE®] (TC, n = 11). Participants supplemented one time daily (480 mg/d) for 10-d including day of exercise up
to 48-h post-exercise. Subjects performed ten sets of ten repetitions at 70 % of a 1-RM back squat exercise. Fastingblood samples, isokinetic MVCs, and quadriceps muscle soreness ratings were taken pre-lift, 60-min, 24-h, and 48-h
post-lift and analyzed by MANOVA with repeated measures.
Results: Muscle soreness perception in the vastus medialis (¼) (p = 0.10) and the vastus lateralis (¼) (p = 0.024) was
lower in TC over time compared to P. Compared to pre-lift, TC vastus medialis (¼) soreness was significantly attenuatedup to 48-h post-lift with vastus lateralis (¼) soreness significantly lower at 24-h post-lift compared to P. TC changes inserum creatinine (p = 0.03, delta p = 0.024) and total protein (p = 0.018, delta p = 0.006) were lower over time and smallerfrom pre-lift levels over time compared to P Significant TC group reductions from pre-lift levels were found for AST and
creatinine 48-h post-lift, bilirubin and ALT 60-min and 48-h post-lift. No significant supplementation effects wereobserved for serum inflammatory or anti-inflammatory markers. None of the free radical production, lipid peroxidation,or antioxidant capacity markers (NT, TBARS, TAS, SOD) demonstrated significant changes with supplementation. Changes
in TC whole blood lymphocyte counts (p = 0.013) from pre-lift were greater compared to P, but TC lymphocyte counts
returned to pre-lift values quicker than P.
Conclusion: Short-term supplementation of Montmorency powdered tart cherries surrounding a single bout of
resistance exercise, appears to be an effective dietary supplement to attenuate muscle soreness, strength decrement
during recovery, and markers of muscle catabolism in resistance trained individuals.
Keywords: Tart cherry, Resistance exercise, Recovery, Antioxidants, Anti-inflammatory, Muscle damage* Correspondence: rkreider@hlkn.tamu.edu
1
Department of Health and Kinesiology, Exercise and Sport Nutrition
Laboratory, Texas A&M University, College Station, TX 77843-4243, USA
Full list of author information is available at the end of the article© 2015 Levers et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

BackgroundA large volume, high intensity strength training workout
activates a load-induced stress response characterized bystructural muscle damage, oxidative stress, and inflammation that facilitates the release of intramuscular proteins into systemic circulation that are usually associatedwith cardiovascular dysfunction, invasive surgery, anddisease [1–4]. The repetitive nature of muscle contractions during bouts of high intensity exercise will induce
muscular injury as a result of ultrastructural disruptions[3, 5–7] that ultimately lead to a muscular repair sequence of events: degeneration, inflammation, regeneration, and fibrosis [3, 8]. Muscle soreness following
exercise is not the direct result of inflammation, but rather a product of high nociceptor and mechanoreceptor
sensitivity to the potent chemicals and by-products released during muscular degeneration [5, 9, 10]. Millions
of people, including athletes, use non-steroidal antiinflammatory drugs (NSAIDs) to help reduce pain andinflammation [3, 5]. NSAID use mitigates the inflammatory
response via non-specific inhibition of the cyclooxygenase(COX-1 and COX-2) enzymes that regulate production of
inflammatory-stimulating prostaglandins [3, 5, 11]. However, NSAIDs remain controversial as some research has
demonstrated that muscle protein synthesis and the function of satellite cells in skeletal muscle hypertrophy arecompromised when COX enzymes are inhibited [12–16],
while other studies have found no NSAID effect on postexercise anabolic processes within muscle [17–19].
Due to the debate surrounding the use of NSAIDs in sportand exercise applications, performance nutrition research
has more recently shifted focus toward phytochemicalcontaining fruits and other functional foods that seem toprovide a beneficial anti-inflammatory and antioxidant effect [20, 21]. A wide variety of antioxidant and polyphenolcontaining functional foods such as purple sweet potatoes[22–24], beet root juice [25–27], cranberries [28, 29], andblueberries [30, 31] have verified health, performanceenhancing, and exercise recovery benefits. However, compared to other functional foods, the high anthocyanin
content of both tart (e.g. Montmorency cherries) and sweet(e.g. Bing cherries) [21, 32–34] have proven beneficial
in health [35–38], inflammatory-related disease states(e.g. cardiovascular disease, diabetes, osteoarthritis,gout) [35, 36, 38–40], and sleep quality [41, 42]. Clinical supplementation success with cherries, particularly
tart cherry whole fruit, concentrates and cultivar juice
blends, spurred an increase in exercise-based research
to prove beneficial effects in mitigating muscle damage,oxidative stress, inflammation, and muscle pain with animpetus to increase performance [21, 43, 44].
We are aware of only two studies that have evaluatedthe effects of tart cherry supplementation on responses toresistance-based exercise. The first study examined 8-d ofPage 2 of 23

tart cherry cultivar-blended juice supplementation on exercise-induced muscle pain surrounding a bout of 40 maximal eccentric elbow flexion contractions (2 sets × 20
repetitions) [20]. As a result of tart cherry supplementation,
strength losses and exercise-induced muscle pain were attenuated compared to a placebo over 4-d of maximal
eccentric elbow flexion exercise [20]. No hematologicalanalysis was performed to decipher potential physiologicaleffects of supplementation. A second study examined theeffects of 10-d tart cherry concentrate supplementation on
physiological markers of muscle damage, oxidative stress,
inflammation, muscle function, and muscle pain surrounding an intensive single-leg 100 repetition knee extensor
training session (10 sets × 10 repetitions) [4]. Similar to thefirst study, functional recovery of isometric muscle strength
was greater as a result of tart cherry supplementation compared to placebo [4]. Hematological analysis demonstrated
reduced oxidative stress following the leg extension protocol that supported the beneficial functional recovery resultsin the tart cherry trial [4]. Interestingly, a recent endurancebased study with blended Montmorency tart cherry juice
supplementation evaluating stress, inflammation, and upper
respiratory symptoms following a marathon run demonstrated an attenuation of post-race incidence and severity ofupper respiratory tract symptoms and inflammation (serum
CRP) [45]. The primary aim of the current study was todetermine if this powdered supplement derived from tart
cherry skins would promote similar attenuation of muscle
soreness, strength losses, and oxidative damage as the tart
cherry cultivar-blended juice and juice concentrate supplemented in the two previous resistance exercise studies. The
secondary objective of this study was to determine whether
short-term (10-d) ingestion of the powdered tart cherry formulation prior to and following a lower body strength exercise protocol of high volume and intensity will effectively
reduce markers of muscle damage, muscle soreness, inflammation, oxidative stress, and attenuate strength losses during subsequent exercise performance.

Methods
Subjects

Twenty-three healthy, resistance-trained males (20.9 ±2.6 yr, 81.7 ± 10.3 kg 14.2 ± 5.4 % body fat, 63.9 ± 8.6 kg
FFM) participated as subjects in this study. Subjects
were recruited through paper and electronically distributed study advertisements at Texas A&M University.
Entrance criteria required subjects to have been involvedin a progressive resistance training program that included regular squat exercise for at least 6 months prior
to study recruitment and be able to perform a standard
barbell back squat in a Smith machine rack of at least
1.5 times their body weight. Proper performance of barbell back squat technique was determined by a National
Strength and Conditioning Association (NSCA) Certified

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Strength and Conditioning Coach (CSCS) during baseline testing. Figure 1 demonstrates the breakdown of thesubject population as it pertains to the study progression. Discontinuation of any subject participation was
not related to any aspect of the supplementation or testing protocol.
All subjects signed informed consent documents andthe study was approved by the Texas A&M University
Institutional Review Board prior to any data collection.
Subjects were not allowed to participate in this study ifthey reported any of the following: 1) any metabolic disorders or taking any thyroid, hyperlipidemic, hypoglycemic,
anti-hypertensive, anti-inflammatory (e.g. NSAIDs), and/or androgenic medications; 2) history of hypertension,
hepatorenal, musculoskeletal, autoimmune, and/or neurological disease(s); 3) allergy to cherries or any cherry components (e.g. polyphenols, anthocyanins, anthocyanidins).
Experimental design

Figure 2 shows the experimental design used in this study.
The study was conducted in a randomized, double-blind,and placebo-controlled manner. All subjects eligible to participate in the study completed a morning familiarization(FAM) session where they were provided detailed information regarding the study design, testing procedures, andsupplementation protocols. Informed consent, medical
history, and endurance training history questionnaires were

Page 3 of 23

also completed during the FAM session. A research nurse
reviewed medical history documents and performed aphysical exam on each subject to ensure safety and participation eligibility. A fasting blood sample was taken at theend of the FAM session if the subject met entrance criteria.
Subjects were asked to not change their dietary habits inany way throughout the study (upon start of supplementation). This was monitored by subject documentation of
dietary intake for 4-d (3 weekdays and 1 weekend day) of
the first seven supplementation days prior to the resistance
exercise challenge. Approximately 10-d prior to the resistance exercise intervention, eligible subjects returned to thelab for a morning baseline testing session to determine
body weight, height, and body composition. The subjects
then were familiarized with the maximal voluntary contraction (MVC) protocol using their dominant leg on an
isokinetic knee extension dynamometer followed by a 5min recovery before determination of their barbell backsquat 1-repetition maximum (1-RM) in a Smith machine rack. Following baseline measurements, subjects
were matched based on relative maximal back squat
strength, fat free mass, body weight, and age accompanied
by a random separation into two groups: 1) a placebogroup or 2) a powdered tart cherry group.
Subjects were instructed to begin supplementation 7-dprior to the resistance exercise challenge (Day 0). Subjects were asked to fast overnight for 10-h to accountFig. 1 Consort diagram breakdown of the subject population as it pertains to the various stages of study progression

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Page 4 of 23

Fig. 2 Experimental study design. DEXA = dual-energy X-Ray absorptiometer, MVC = maximal voluntary contraction, 1-RM = 1-repetition maximum,
NSAID = non-steroidal anti-inflammatory drugs, GPRS = graphic pain rating scale, 7-d = 7-day, 48-h = 48-hourfor diurnal variation as well as refrain from exercise andconsumption of NSAIDs for 48-h prior to all testingdays. Due to the large variety of NSAIDs commonly
available with half-life data ranging from approximately
1.6 to 30-h [46], the 48-h restriction was implemented
to help reduce any potential concurrent effects. Exercise
logs were not implemented to track subject activity patterns over the study protocol. On the day of the resistance exercise challenge, subjects reported to the lab
where body weight, resting heart rate, and resting bloodpressure were measured. Subjects then donated a fasting
venous blood sample (approximately 20 ml) using standard clinical procedures and rated perceptions of muscle
soreness to a standardized application of pressure on
their dominant thigh at three designed locations using agraphic pain rating scale (GPRS). The subjects thencompleted the MVC protocol using their dominant leg
on an isokinetic knee extension dynamometer followed
by a 5-min recovery before the barbell back squat protocol utilizing a Smith machine rack was completed with aTendo power and speed analyzer attached to the bar.
Fasting (except 60-min post-lift) blood samples, GPRS
ratings of quadriceps muscle soreness, and the isokinetic
leg extension MVC protocol were completed at 60-min,24-h and 48-h of post-lift recovery. The last day of supplementation correlated with 48-h post-lift recovery.
Exercise protocol
Isokinetic knee extension maximal voluntary contractions

At baseline, before the squat exercise, 60-min after thesquat exercise, 24-h and 48-h post-lift exercise, subjects
performed an isokinetic knee extension MVC test using
their dominant leg on a Kin-Kom 125AP Isokinetic

Dynamometer (Chattanooga-DJO Global Inc., Vista, CA,
USA). During the baseline familiarization of the isokinetic leg extension MVC protocol, standard procedures
were used to determine and record proper subject positioning on the Kin-Kom to ensure accurate repeatabilityof the testing protocol across all five sessions. The subject warmed-up on the isokinetic dynamometer by performing three-sets of five repetitions of dominant knee
extension/flexion at 50 % of their MVC (determined
during the FAM) with 1-min recovery between sets.
After the 1-min recovery following the third warm-up
set, the subject performed 3 MVCs in succession withno rest between contractions. Subjects were allotted a 5min recovery between the isokinetic MVC and barbell
back squat protocols. Test to test variability of performing this test yielded average CV values of ±8.47 % with atest retest correlation of r = 0.88.
Barbell back squat 1-repetition maximum determination

At baseline, following the 5-min recovery after the isokinetic MVCs, subjects performed three-sets of five repetitions of a barbell back squat on a Smith machine rack
(Life Fitness, Schiller Park, IL, USA, model SSM) at 50 %of their anticipated 1-RM with 2-min recovery between
sets. Following the warm-up protocol, the barbell back squat
1-RM was determined on the Smith machine rack using astandard 1-RM protocol defined by the NSCA with 2-minrecovery periods between 1- repetition sets of increasing
weight. All 1-RM determinations were made within 3–5 setsbeyond the warm-up. In order to successfully complete eachset, the subject was required to reach thigh parallel depth asdetermined by a NSCA Certified Strength and Conditioning
Coach (CSCS) providing a verbal “up” cue during spotting.

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Barbell back squat exercise protocol

On the day of the resistance exercise challenge, following
the 5-min recovery after the isokinetic MVCs, subjects
performed one warm-up set of ten repetitions of the barbell back squat at 50 % 1-RM (determined during baseline). After 3-min recovery, the subjects performed ten
sets of ten repetitions of a barbell back squat at 70 % 1RM with 3-min recovery between sets. If necessary, thebarbell back squat resistance was adjusted such that eachsubject completed the 100 repetition (10 sets × 10 repetitions) volume to thigh parallel depth, keeping the training
volume consistent across the study population. This
protocol is closely replicated after the repeated leg extension exercise implemented previously by Bowtell etal. [4]. A Tendo Power and Speed Analyzer (TENDOSports Machines, Trencin, Slovak Republic, model
PSA310) was attached to the bar during all 100 repetitions completed during the squat protocol to determine
peak power, average force, and total work for each repetition. Following the squat protocol, subjects were only
permitted to stretch with limited ambulation until the
60-min post-lift testing protocol was completed.
Supplementation protocol

Subjects were assigned in a double-blinded and randomized manner to ingest either a rice flour placebo (P, n = 12)
or powdered tart cherry (TC, n = 11). Subjects werematched into one of the two groups according to relative
maximal back squat strength, fat free mass, body weight,
and age. Subjects were instructed to ingest 480 mg of the
supplements with breakfast at 0800 7-d prior to, the dayof, and 2-d following the resistance exercise challenge fora total supplementation timeline of 10-d. The tart cherry
supplements contained 480 mg of freeze dried Montmorency tart cherry skin powder derived from tart cherryskins obtained after juicing (CherryPURE™ Freeze Dried
Tart Cherry Powder, Shoreline Fruit, LLC, Transverse City,
MI, USA). Prior analytical testing conducted in 2012 by
Atlas Bioscience (Tuscon, AZ, USA) demonstrated that
290 mg of CherryPURE™ provides approximately 600 mg
of phenolic compounds and 40 mg of anthocyanins, whichis equivalent to consuming 10.5 fluid ounces of tart cherry
juice. The supplements were prepared for distribution by
Shoreline Fruit, LLC and sent to Advanced Laboratories(Salt Lake City, UT, USA) to quantify total phenolic compound and anthocyanin content of the powdered tart
cherry supplements. Supplements were prepared in standardized color capsules and packaged in generic bottles by
Shoreline Fruit, LLC for double blind administration.
Procedures
Dietary inventories

Within the first 7-d of supplementation, subjects were
instructed to record all food and fluid intake over a 4-dPage 5 of 23

period (3 weekdays, 1 weekend day), which was reflective of their normal dietary intake. Dietary inventories
were then reviewed by a registered dietician and analyzed for average energy, macronutrient, and dietary
antioxidant intake using ESHA Food Processor (Version
8.6) Nutritional Analysis software (ESHA Research Inc.,
Salem, OR, USA).
Anthropometrics and body composition

At the beginning of every testing session, subjects had
their height and body mass measured according to standard procedures using a Healthometer Professional 500KL(Pelstar LLC, Alsip, IL, USA) self-calibrating digital scalewith an accuracy of ± 0.02 kg. Whole body bone densityand body composition measures (excluding cranium) were
determined with a Hologic Discovery W Dual-Energy Xray Absorptiometer (DEXA; Hologic Inc., Waltham, MA,
USA) equipped with APEX Software (APEX Corporation
Software, Pittsburg, PA, USA) by using procedures previously described [47, 48]. Mean test-retest reliability studies
performed on male athletes in our lab with this DEXA
machine have revealed mean coefficients of variation for
total bone mineral content and total fat free/soft tissue
mass of 0.31–0.45 % with a mean intraclass correlation of
0.985 [49]. On the day of each test, the equipment wascalibrated following the manufacturer’s guidelines.
Muscle soreness perception measurement

Pressure application to the three specified areas of thequadriceps muscle group on the subject’s dominant leg
was standardized to 50 N of pressure using a handheld
Commander Algometer (JTECH Medical, Salt Lake City,
UT, USA). Force was applied to each site of muscle contact with a cylindrical metal probe through a 1 cm diameter head. The standard amount of pressure was applied
to the vastus lateralis (VL) at both 25 and 50 % of the
distance between the superior border of the patella andthe greater trochanter of the femur at the hip and to thevastus medalis (VM) at 25 % of the distance between the
aforementioned landmarks. These three specific locations were measured and marked with a permanentmarker on each subject during the initial muscle soreness perception measurement before the resistance exercise challenge. The participants were asked to maintainthese three marked locations between testing sessions to
avoid error with secondary measurement. The subject
was seated in a reclined supine position and given the
GPRS sheet to evaluate the perception of muscle soreness at each of the three quadriceps locations. The orderof pressure application was standardized across all sessions and participants: 25 % VM, 25 % VL, and 50 % VL.
The 50 N of pressure was applied to a relaxed quadriceps
at each of the three locations using the algometer for aperiod of 3-sec to give the subject enough time to record

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

their soreness evaluation on the GPRS. Perceptions ofmuscle soreness were recorded by measuring the distance (centimeters) of the participant mark on the GPRS
from 0 cm (no pain). Test-to-test reliability for thisprotocol revealed a mean intraclass correlation of 0.90.

Page 6 of 23

levels of control fluids purchased from the manufacturer
to calibrate acceptable SD and CV values for all aforementioned assays. Samples were re-run if the observed
values were outside control values and/or clinical norms
according to standard procedures.

Blood collection

Subjects were required to fast (except 60-min post-lift)
for 10-h prior to donating approximately four teaspoons(20 mL) of venous blood from an antecubital vein using
standard phlebotomy procedures at each of the testing
sessions. Blood analyzed for markers of muscle damage,oxidative stress, inflammation, and clinical chemistry
panels were collected in two 7.5 mL BD Vacutainer®
serum separation tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), left at room temperaturefor 15-min, and then centrifuged at 3500 rpm for 10min using a standard, refrigerated (4 °C) bench top
Thermo Scientific Heraeus MegaFuge 40R Centrifuge(Thermo Electron North America LLC, West Palm Beach,FL, USA). Serum supernatant was removed and storedat −80 °C in polypropylene microcentrifuge tubes forlater analysis. Blood was also collected in a single 3.5 mL
BD Vacutainer® lavender top tube containing K2 EDTA
(Becton, Dickinson and Company, Franklin Lakes, NJ,
USA), left at room temperature for 15-min, and refrigerated for approximately 3–4 h before subsequent
complete blood count analysis.
Clinical chemistry analysis

Whole blood samples were analyzed for complete bloodcount with platelet differentials (hemoglobin, hematocrit,
red blood cell counts (RBC), white blood cell counts(WBC), lymphocytes, granulocytes (GRAN), and midrange absolute count (MID)) using an Abbott Cell Dyn
1800 (Abbott Laboratories, Abbott Park, IL, USA) automated hematology analyzer. Internal quality control for
the Abbott Cell Dyn 1800 was performed using threelevels of control fluids purchased from the manufacturer
to calibrate acceptable standard deviation (SD) and coefficients of variation (C V) values for all aforementioned
whole blood cell counts. Serum samples were analyzed
using a Cobas c111 (Roche Diagnostics GmbH, Indianapolis, IN, USA) automated clinical chemistry analyzer
that was calibrated and optimized according to manufacturer guidelines. This analyzer has been known to behighly valid and reliable in previously published reports[50]. Each serum sample was assayed for a standard partial metabolic panel [(aspartate aminotransferase (AST),
alanine aminotransferase (ALT), and total bilirubin)] andclinical markers of protein and fatty acid metabolism[(uric acid, creatinine, blood urea nitrogen (BUN), total
protein, and creatine kinase (CK)]. The internal quality
control for the Cobas c111 was performed using two

Markers of anabolic/catabolic hormone status

Serum samples were assayed using standard commercially available enzyme-linked immunosorbent assay kits
(ELISAs) for cortisol and testosterone (ALPCO Diagnostics, Salem, NH, USA) hormone analysis. Serum concentrations were determined calorimetrically using a BioTek
ELX-808 Ultramicroplate reader (BioTek Instruments
Inc., Winooski, VT, USA) at an optical density of 450 nm
against a known standard curve using manufacturer recommended procedures. Samples were run in duplicate
according to standard procedures to ensure validity ofmeasurement. Test to test variability of performing theseassays yielded average CV values for the aforementioned
markers of: CORT (±6.85 %), and TEST (±4.47 %) witha test retest correlation for the same markers of: CORT
(r = 0.92), TEST (r = 0.98).

Markers of oxidative stressSerum samples were assayed using standard commercially available ELISA kits for Superoxide Dismutase(SOD Activity Assay kit), Total Antioxidant Status (TAS,
Antioxidant Assay kit), Thiobarbituric Acid Reactive Substance (TBARS, Malondialdehyde-MDA, TCA methodkit) (Cayman Chemical Company, Ann Arbor, MI, USA),and Nitrotyrosine (ALPCO Diagnostics, Salem, NH, USA).
Serum concentrations for SOD and Nitrotyrosine were
determined calorimetrically using the previously statedinstrumentation at the same optical density (450 nm)
against a known standard curve using standard procedures. Serum concentrations for TAS were also determinedcalorimetrically using the aforementioned instrumentation
at an optical density of 405 nm against a known standard
curve using standard procedures. Lastly, serum concentrations for TBARS were also determined fluorometricallyusing a SpectraMax Gemini multimode plate reader(Molecular Devices LLC, Sunnyvale, CA, USA) at an excitation wavelength of 530 nm and an emission wavelength of 550 nm against a known standard curve using
standard procedures. Samples were run in duplicate according to standard procedures to ensure validity ofmeasurement. Test to test variability of performing
these assays yielded average C V values for the aforementioned markers of: SOD (±8.35 %), TAS (±14.24 %),TBARS (±8.30 %), and NT (±10.03 %) with a test retest
correlation for the same markers of: SOD (r = 0.83),
TAS (r = 0.85), TBARS (r = 0.94), and NT (r = 0.99).

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Cytokine/Chemokine markers of inflammation

Serum markers of inflammation [(interleukin-1β (IL-1β),IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13,
tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ),
and granulocyte-macrophage colony-stimulating factor(GM-CSF)] were measured by using a commerciallyavailable Milliplex MAP 13-Plex Human High Sensitivity
T-Cell Magnetic Bead Panel kit (EMD Millipore Corporation, St. Charles, MO, USA) that was optimized for human serum samples. A minimum of 100 positive beads
for each cytokine/chemokine was acquired with a Luminex MagPix instrument (Luminex Corporation, Austin,
TX, USA). This instrument has been proven to be highly
valid and reliable in previously published reports across
many disciplines [51–54]. Manufacturer supplied controls were used to monitor the coefficients of variation.
Samples were run in duplicate according to standard
procedures to ensure validity of measurement. Test to
test variability of performing these assays yielded average
CV values for the aforementioned markers of: IL-1β(±4.37 %), IL-2 (±5.52 %), IL-4 (±4.59 %), IL-5 (±4.30 %),IL-6 (±4.26 %), IL-7 (±5.05 %), IL-8 (±4.55 %), IL-10(±5.05 %), IL-12p70 (±4.92 %), IL-13 (±4.99 %),TNF-α(±6.05 %), IFN-γ (±4.42 %), and GM-CSF (±5.22 %) witha test retest correlation for the same markers of: IL-1β(r = 0.98), IL-2 (r = 0.99), IL-4 (r = 0.99), IL-5 (r = 0.99), IL-6
(r = 0.99), IL-7 (r = 0.99), IL-8 (r = 0.99), IL-10 (r = 1.00),
IL-12p70 (r = 1.00), IL-13 (r = 0.99), TNF-α (r = 0.95),
IFN-γ (r = 1.00), and GM-CSF (r = 1.00).
Statistical analysis

Individual group and time data are presented throughout
the text and in all tables as means (± SD), while groupeffects are presented as means (± SEM). All related variables were grouped and analyzed using repeated measures MANOVA in IBM SPSS Statistics Software version
22.0 for Windows (IBM Corporation, Armonk, NY, USA)to assess values observed and changes from pre-liftlevels in response to the supplement administered. Posthoc LSD pairwise comparisons were used to analyze any
significance among groups where needed with Cohen’s dcalculations employed to determine effect magnitude.
Data was considered statistically significant when theprobability of error was less than 0.05 and considered to
be trending when the probability of error was less than
0.10. Statistical trends (p < 0.05 to p < 0.10) were noted
as is common practice in studies with relatively small
sample size [55].

Results
Subject baseline characteristics

A total of 23 healthy, resistance-trained men completed
the protocol for this study. Participants were 20.9 ±2.6 years, 81.7 ± 10.3 kg, 14.2 ± 5.4 % body fat, and 63.9 ±Page 7 of 23

8.6 kg fat free mass. Participant demographic data are presented in Table 1. One-way ANOVA revealed no significant
differences (p > 0.05) in baseline demographic markers. No
significant differences in back squat 1-RM (p = 0.70) andrelative squat strength ratio (p = 0.85) were reported acrossgroups, hence the groups were generally well matched.
One-way ANOVA analysis also demonstrated no differences in total work performed (p = 0.79) across groups, indicating that similar work was performed during theexercise intervention and differences can likely be attributed to the nutritional intervention.
Nutritional intake and complianceNutritional intake was monitored over a 4-d period
within the first 7-d of supplementation. Relevant nutritional components analyzed are listed in Table 2. No statistically significant interactions were observed acrossgroups with respect to dietary intake.
Muscle soreness perception assessment

Table 3 presents perceptions of muscle soreness acrossthe resistance exercise protocol. The overall MANOVA
analysis revealed a significant overall Wilks’ Lambda
time (p < 0.001) interaction and a non-significant overall
group x time effect (p = 0.20). Perception of muscle soreness in all three muscle testing locations irrespective ofgroup significantly increased over time, peaking 48-h
post-lift, indicating the onset of muscle soreness as aresult of the lifting protocol. A significant difference
between groups over time was found in vastus lateralis(¼) soreness perception (p = 0.024) where ratings increased by 55–170 % from pre-lift values in P, but only
35–104 % in TC over the recovery. Post-hoc analysis indicated a significantly attenuated increase in muscle
soreness perception 24-h post-lift for TC supplementing
subjects compared to P (see Fig. 3). Effects of supplementation tended to be different in the perception of
vastus medialis (¼) soreness (p = 0.10) with significantly
lower muscle soreness in TC versus P up to 48-h postlift (see Fig. 3). Soreness perception ratings in the vastus
medialis (¼) increased by 28–83 % from pre-lift values
in P, but ranged from a 6 % decrease to only a 58 %increase in TC over the recovery.
Isokinetic maximal voluntary contraction performanceassessment

Table 4 reports the total work performed during the 3repetition isokinetic flexion/extension MVC test. The
overall MANOVA analysis revealed both a non-significant
Wilks’ Lambda time interaction (p = 0.96) and a nonsignificant group x time effect (p = 0.75). The 3-repetition
work summation univariate measures for extension (p <0.001), flexion (p = 0.022), and total (p < 0.001) demonstrated significant changes over time, with the lowest work

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Page 8 of 23

Table 1 Demographics by Study Group
Variable

Group

Mean

Group (SEM)p-value

N

P

12

n/a

n/a

TC

11

n/a

Total

23

n/a

Age

Height (cm)Weight (kg)Baseline HR (bpm)2

BMD (g/cm )LM (kg)

FFM (kg)

FM (kg)Body Fat (%)

Back Squat 1-RM (lbs)

Relative Squat Strength Ratio

Workout Total Work (kJ)P

20.58±1.78

0.51

TC

21.18±3.34

1.01

Total

20.87±2.60

0.54

P

177±5.28

1.52

TC

178±10.71

3.23

Total

177±8.17

1.70

P

82.62±7.39

2.13

TC

80.64±13.16

3.97

Total

81.67±10.35

2.16

P

61.17±10.40

3.00

TC

66.09±6.67

2.01

Total

63.52±8.98

1.87

P

1.16±0.10

0.03

TC

1.12±0.14

0.04

Total

1.14±0.12

0.02

P

61.41±4.87

1.41

TC

61.55±11.16

3.36

Total

61.48±8.27

1.73

P

63.83±5.06

1.46

TC

63.92±11.64

3.51

Total

63.87±8.62

1.80

P

11.81±5.44

1.57

TC

14.10±13.58

4.09

Total

12.90±10.00

2.08

P

15.31±6.27

1.81

TC

13.09±4.31

1.30

Total

14.25±5.42

1.13

P

318±6218.03

TC306±82

24.72

Total

313±7114.81

P1.75±0.27

0.08

TC

1.73±0.34

0.10

Total

1.74±0.30

0.06

P

0.68±0.14

0.04

TC

0.70±0.23

0.07

Total

0.69±0.18

0.04

0.593

0.651

0.659

0.195

0.389

0.970

0.981

0.595

0.338

0.696

0.855

0.798

Mean data expressed as means ± SD
HR heart rate, BMD bone mineral density, LM lean mass, FFM free-fat mass, FM fat mass, 1-RM 1-Repetition maximum

attained 60-min post-lift in all three measures. No aspectof the isokinetic strength recovery work performed demonstrated a significant group effect over time. Whilechanges from pre-lift were not statistically significant

between groups across time, the drop in 3-repetition summation of flexion (p = 0.21; d = 0.45), extension (p = 0.23;
d = 0.45), and total work (p = 0.15; d = 0.55) performed 60min post-lift may have been attenuated in TC comparedLevers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Page 9 of 23

Table 2 Dietary Analysis by Study Group
Variable

Group

Mean

Group (SEM)p-value

Average Daily Caloric Consumption (kcal)

P

2617±721

208

0.605

TC

2455±753

227

Total

2540±724

151P

147.86±43.13

12.45

TC

153.06±100.39

30.27

Dietary Protein (g)Dietary Carbohydrates (g)Dietary Fat (g)Dietary Beta-Carotene (mcg)

Dietary Vitamin C [Ascorbic Acid] (mg)

Dietary Vitamin E [Alpha-Tocopherol] (mg)Total

150.35±74.28

15.49

P

244.47±62.58

18.07

TC

223.52±70.90

21.38

Total

234.45±66.01

13.76

P

84.54±23.55

6.80

TC

95.23±38.55

11.62

Total

89.65±31.35

6.54

P

2954±3436

992

TC

2111±5260

1586

Total

2551±4320

901

P

80.79±65.99

19.05

TC

50.56±38.41

11.58

Total

66.33±55.55

11.58

P

5.91±6.04

1.74

TC

6.24±7.44

2.24

Total

6.07±6.59

1.37

0.871

0.460

0.427

0.651

0.199

0.910

Mean data expressed as means ± SD

to P based upon the moderate to large Cohen’s d effectsize calculations. Average recovery decreases in flexion,
extension, and total work performance from pre-lift values
were 5, 19, and 14 % respectively in P, but only 2, 16, and
10 % in TC.

Markers of mechanical damage and physiological stress

Table 5 presents the serum mechanical damage andphysiological stress markers tested in the standard clinicalsafety panel. The overall MANOVA analysis revealed asignificant overall Wilks’ Lambda time (p < 0.001) effect,

Table 3 Quadriceps Muscle Soreness Perception
Variable
Algo I (cm)

Algo II (cm)Group

60-min Post

24-hr Post

48-hr Post

Group Meanp-value (GG)

p-value (WSC)P

4.34±2.52

5.53±3.15

7.78±2.93

7.94±2.92

6.40±0.76

G=0.139TC

3.80±2.86

3.59±2.76

5.50±2.68

5.99±3.59

4.72±0.79

T<0.001*

TL<0.001*

G X T=0.236

G X Tq=0.172

Ψ◊Ψ◊Time Mean

4.07±0.56

4.56±0.62

6.64±0.59

6.96±0.68P

2.44±1.60

3.76±2.24Ψ

6.60±2.99^Ψ◊6.20±3.64Ψ◊

4.75±0.69

G=0.773TC

2.98±2.70

4.03±2.93Ψ

4.76±3.03Ψ

6.07±3.67Ψ◊#

4.46±0.72

T<0.001*

TL<0.001*

2.71±0.46ΨΨ◊

6.14±0.76Ψ◊G X T=0.187

G X Tq=0.024*

Time Mean
Algo III (cm)

Pre-Lift3.90±0.54

5.68±0.63P

3.25±2.65

3.42±3.24

6.55±3.80

6.18±3.36

4.85±0.81

G=0.609TC

2.68±2.67

3.76±3.18

4.89±3.87

5.64±3.74

4.24±0.85

T<0.001*

TL<0.001*

G X T=0.427

G X TL=0.690

Time Mean

2.96±0.55

3.59±0.67Ψ◊

5.72±0.80

Ψ◊5.91±0.74

Individual group and time data expressed as means ± SD, while group effects are presented as means ± SEM. Data represents the participant soreness perception
in the quadriceps muscle group at each test session during the 10 day intervention. MANOVA analysis revealed overall Wilks' Lambda time (p<0.001) and group xtime (p=0.199). Univariate ANOVA p-levels from MANOVA analysis are presented for each variable. Univariate ANOVA p-levels are listed first by the GreenhouseGeisser (GG) analysis and then by the within-subjects contrasts (WSC) to demonstrate the potential shape of the time or group x time interaction with significanceindicated by the following super/subscripts: * indicates p<0.05 p-level significance, § indicates p<0.10 p-level significance. LSD post hoc analysis is indicated by
the following superscripts: ^ represents p<0.05 difference between groups, Ψ represents p<0.05 difference from pre-lift, ◊ represents p<0.05 difference from 60min post, # represents p<0.05 difference from 24-hr post. Algo I = Algometer location #1: Vastus Medalis 1/4; Algo II = Algometer location #2: Vastus Lateralis 1/4;
Algo III = Algometer location #3: Vastus Lateralis 1/2; G = group p-level; T = time p-level; G x T = interaction; q = quadratic p-level; L = linear p-level

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Page 10 of 23

Fig. 3 Perceptions of muscle soreness. Data expressed as means ± SE and significance indicated by the following super/subscripts: * indicates p < 0.05
p-level significance, § indicates p < 0.10 p-level significance. LSD post hoc analysis is indicated by the following superscripts: ^ represents p < 0.05 difference
between groups, Ψ represents p < 0.05 difference from pre-lift, ◊ represents p < 0.05 difference from 60-min post, # represents p < 0.05 difference from24-h post

Table 4 Isokinetic Maximal Voluntary Contraction Knee Extension/Flexion Total Work Performance
Variable
IK Total Ext Work (N)Group

Baseline

60-min Post 24-hr Post

48-hr Post

Group Mean p-value (GG) p-value (WSC)P

1791±2071682±235

1278±275

1408±296

1387±291

1509±90G=0.870TC

1757±378

1612±437

1298±422

1418±463

1352±383

1487±94

T<0.001*

TL<0.001*

Time Mean 1774±295†

1648±340

1288±345

1413±376

1370±331

G X T=0.792

G X Tq=0.662

971±126^ 962±141^

913±174^925±153^910±113^†936±58G=0.1811073±252

1064±225

1058±305

1010±289†

1053±61

T=0.087§

TL=0.022*

G X T=0.663

G X Tq=0.206

IK Total Flex Work (N) P
TCTime Mean 1020±199
IK Total Work (N)Pre-Lift1060±277
1009±218†Ψ

986±210

†Ψ◊

†Ψ

†Ψ

989±242958±217P2762±293

2644±354

2191±388

2333±407

2296±373

2445±141

G=0.646TC

2829±611

2673±687

2363±603

2476±733

2362±644

2540±148

T<0.001*

TL<0.001*

Time Mean 2794±462†

G X T=0.692

G X Tq=0.470

2658±527†Ψ

2273±498†Ψ◊

2401±577†Ψ

2328±509

Individual group and time data expressed as means ± SD, while group effects are presented as means ± SEM. Data represents the isokinetic MVC knee extension
and flexion performance each test session during the 10 day intervention. MANOVA analysis revealed overall Wilks' Lambda time (p<0.960) and group x time
(p=0.748). Univariate ANOVA p-levels from MANOVA analysis are presented for each variable. Univariate ANOVA p-levels are listed first by the Greenhouse-Geisser(GG) analysis and then by the within-subjects contrasts (WSC) to demonstrate the potential shape of the time or group x time interaction with significanceindicated by the following super/subscripts: * indicates p<0.05 p-level significance, § indicates p<0.10 p-level significance. LSD post hoc analysis is indicated by
the following superscripts: ^ represents p<0.05 difference between groups, † represents p<0.05 difference from baseline value, Ψ represents p<0.05 difference
from pre-lift, ◊ represents p<0.05 difference from 60-min post, # represents p<0.05 difference from 24-hr post. Ext = extension; Flex = flexion; IK = isokinetic; MVC= maximal voluntary contraction; G = group p-level; T = time p-level; G x T = interaction; q = quadratic p-level; L = linear p-level

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

but no difference between groups over time (p = 0.50).
Univariate measures for uric acid (p < 0.001), total bilirubin (p = 0.026), creatinine (p = 0.003), and total protein(p < 0.001) demonstrated significant changes over time,
peaking 60-min post-lift. CK (p = 0.025) and AST (p =0.003) also significantly changed over time, peaking 24-h
post-lift. Significant group differences over time and groupby time changes from pre-lift were reported for creatinine(p = 0.030, delta p = 0.024) and total protein (p = 0.018,
delta p = 0.006). Analyzing the change from pre-liftlevels, significant differences across groups for creatinine(p = 0.007) and total protein (p = 0.004) were also evident.
Serum creatinine increased on average 2 % over pre-lift
values during the recovery in P, but actually decreased 7 %below pre-lift in TC. Serum total protein increased 2–9 %over pre-lift values during the recovery in P, but decreased
0–4 % below pre-lift in TC. Subsequent post-hoc analysis
indicated a significantly attenuated TC creatinine response
48-h post-lift and a TC total protein level that never differed from pre-lift measures 60-min and 48-h post-lift
compared to deviations in P (see Fig. 4). Increases in serum
CK levels tended to be lower in TC over time compared to
P (p = 0.10). Serum CK levels increased on average 135 %over pre-lift values during the recovery in P, but increased
only 98 % in TC. No other significant differences between
groups over time or deviations from normal human clinicalranges were observed for kidney/liver enzymes [e.g. AST(p = 0.16), ALT (p = 0.27)] or markers of protein catabolism [bilirubin (p = 0.12), BUN:creatinine ratio (p =0.39), uric acid (p = 0.15)]. However, further post-hoc
analyses revealed TC facilitated post-lift attenuations inbilirubin, AST, and ALT 48-h post-lift compared to P
(see Figs. 4 and 5).
Anabolic/catabolic hormone response markers

Table 6 shows the serum anabolic and catabolic hormone
markers analyzed in response to exercise. The overall
MANOVA analyses revealed both non-significant overall
Wilks’ Lambda time (p = 0.29) and overall group changes
across time (p = 0.46). Cortisol levels tended to changeover time (p = 0.093). Serum cortisol levels were significantly different between groups over time (p = 0.029) andrevealed how supplementation caused significant variability (p = 0.032) in cortisol changes from pre-lift levels
across the recovery period. Serum cortisol levels increasedon average 13 % over pre-lift values during the recovery inP, but only 8 % in TC. The delta post-hoc analysis revealed
that TC cortisol levels were not different from pre-liftlevels and were significantly lower at 48-h post-lift compared to rising cortisol levels in P (see Fig. 5).
Markers of free radical production and oxidative stress

Table 7 reports the serum markers of free radical production [reactive oxygen species (ROS) and reactive nitrogen

Page 11 of 23

species (RNS)] analyzed in response to exercise. The overall MANOVA analysis revealed no significant overall
Wilks’ Lambda time (p = 0.32) interaction and no overall
group differences over time (p = 0.76). None of the univariate measures for markers of free radical production,
lipid peroxidation, and antioxidant capacity/activity (NT,TBARS, TAS, and SOD) reported main time effects or differences between groups over time.
Inflammatory response markers

Table 8 shows the serum inflammatory cytokine and chemokine markers analyzed in response to exercise. The
overall MANOVA analysis revealed a significant overall
Wilks’ Lambda time (p < 0.001) interaction, but a nonsignificant overall group x time effect (p = 0.30). Univariate
measures for TNF-α (p = 0.001), IL-1β (p = 0.030), IL-6
(p = 0.023), and IL-8 (p = 0.018) demonstrated significant changes over time and from baseline measures,
peaking 24-h post-lift. No significant differences between
groups were observed over time for any of the inflammatory cytokines or chemokines.
Anti-inflammatory response markers

Table 9 presents the serum anti-inflammatory cytokine
markers analyzed in response to exercise. The overall
MANOVA analysis revealed a significant overall Wilks’Lambda time (p < 0.001) interaction, but a non-significant
overall effect of treatment over time (p = 0.45). Univariate
measures for IL-4 (p = 0.001) and IL-7 (p = 0.033) reported
significant main time effects with IL-13 levels approachingsignificance across time (p = 0.055), peaking 60-min postlift. No significant group effects over time were reported
for any of the anti-inflammatory cytokine markers.
Clinical markers of immune-related complete bloodcountsTable 10 displays immune response-related complete
blood count markers analyzed in response to exercise.
The overall MANOVA analysis demonstrated significant
overall Wilks’ Lambda time interaction (p < 0.001), but anon-significant overall difference between groups overtime (p = 0.68). Univariate measures for lymphocytes(p < 0.001) and MID (p < 0.001) demonstrated significant changes over time and from baseline measures,
most depressed 60-min post-lift. WBC (p = 0.004) and
GRAN (p = 0.003) also significantly changed over time,with lowest values 48-h post-lift. MID levels tended tobe different between groups over time (p = 0.062). Significant group changes over time (p = 0.015) and differences ingroup effects from pre-lift levels over the recovery period(p = 0.013) were reported for lymphocytes. Lymphocyte
levels decreased on average 23 % below pre-lift values during the recovery in P, but only 11 % in TC. Subsequentpost-hoc analysis showed a significantly greater lymphocyte

Variable

Group

Baseline

Pre-Lift

60-min Post

24-hr Post

48-hr Post

Group Meanp-value (GG)

AST (U/L)P

31.85±9.65

27.47±6.40

30.79±7.88

41.39±13.31

37.67±10.33

33.83±2.19

G=0.919TC

37.70±17.65

28.39±7.62

29.53±6.46

40.59±17.59

31.32±11.57

33.51±2.29

T=0.003*

Tq=0.759

Ψ◊Ψ#

G X T=0.316

G X TL=0.162

ALT (U/L)

Total Billirubin (umol/L)

Urea/BUN (mmol/L)Time Mean

34.78±2.93

27.93±1.46

P33.81±18.74

32.21±12.26

BUN/Creatinine Ratio

Uric Acid (umol/L)

CK (U/L)

Total Protein (mmol/L)Ψ

30.16±1.51

40.99±3.2334.19±13.60

36.78±14.97

34.50±2.2835.80±13.84

34.56±4.19
29.94±4.38

G=0.455

TC

31.78±13.22

30.01±19.33

28.32±15.83

31.08±19.58

28.54±17.41

Time Mean

32.79±3.4131.11±3.3431.26±3.07

33.93±3.61Ψ◊

32.17±3.27P

8.00±3.67

7.25±3.47

9.08±5.31

8.14±3.82

7.35±3.21

7.96±1.01

G=0.395

TC

9.65±3.85

9.98±4.95

9.58±4.01

9.79±5.20

7.19±3.21

9.24±1.06

T=0.064§

Tq=0.026*

G X T=0.306

G X TL=0.125†Ψ◊#

T=0.529

TL=0.180

G X T=0.521

G X TL=0.266

Time Mean

8.83±0.78

8.61±0.89

9.33±0.99

8.97±0.95

7.27±0.67

P

6.59±1.54

6.16±1.74

6.07±1.59

6.48±1.96

5.92±2.33

6.24±0.44

G=0.794TC

6.49±1.39

6.75±1.30

6.40±1.33

6.59±1.83

5.83±1.66

6.41±0.46

T=0.061§

TL=0.055§

G X T=0.594

G X Tq=0.238

Time Mean
Creatinine (umol/L)†

p-value (WSC)

6.54±0.31

6.46±0.32

6.24±0.31†Ψ#6.53±0.40

5.88±0.43◊

P

96.50±13.23

97.19±19.28

102.76±17.25^

98.91±21.16^

95.20±17.73^

98.11±4.05

G=0.273TC

91.82±11.99

96.84±15.27

95.42±14.37

91.52±11.27

81.99±14.87†Ψ◊#

91.52±4.23

T=0.004*

Tq=0.003*

G X T=0.178

G X TL=0.030*†Ψ◊#Time Mean

94.16±2.64

97.02±3.65

99.09±3.33

95.21±3.58

88.60±3.43

P17.11±4.38

15.97±3.99

14.82±3.66

16.66±5.67

15.50±5.26

16.01±1.08

TC17.65±3.76

17.51±3.83

16.82±3.74

17.85±4.3517.64±3.80

17.50±1.13Time Mean

17.37±4.01

16.71±3.90

15.78±3.75†Ψ

17.23±5.00

16.53±4.64

P

328±66317±66444±136367±80349±88361.12±20.37

G=0.647TC

306±52310±54470±117350±93302±79347.44±21.28

T<0.001*

Tq<0.001*†ΨG X T=0.298

G X Tq=0.146

◊#

†Ψ◊

Time Mean

317±12314±13457±27358±18325±17P

468±288322±243451±267967±700855±981613±82529±86

G=0.353
T=0.214

Tq=0.225

G X T=0.721

G X TL=0.388

G=0.487TC

843±1156259±146355±198770±470415±213Time Mean

656±172291±42†403±49Ψ869±126Ψ◊

635±151Ψ

P

78.67±6.11

67.55±7.96^†

68.67±9.45†

73.67±11.11†Ψ#

72.16±1.52

G=0.30572.43±6.72†

70.66±7.92†Ψ◊

74.47±1.59

T<0.001*70.55±1.72†

TCTime Mean

80.53±4.60
79.60±1.1474.13±2.62†70.84±1.26†72.23±6.47†Ψ†

74.59±4.18†Ψ73.41±1.15†72.17±2.03

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Table 5 Markers of Muscle Catabolism, Secondary Muscle Damage, and Physiological Stress

T=0.025*

Tq=0.222

G X T=0.209

G X TL=0.100§
Tq<0.001*§

G XT=0.091

G X Tq=0.018*

Page 12 of 23

Individual group and time data expressed as means ± SD, while group effects are presented as means ± SEM. Data represents the response to muscle catabolism, mechanical damage, and physiological stress at eachtest session during the 10 day intervention. MANOVA analysis revealed overall Wilks' Lambda time (p<0.001) and group x time (p=0.504). Univariate ANOVA p-levels from MANOVA analysis are presented for eachvariable. Univariate ANOVA p-levels are listed first by the Greenhouse-Geisser (GG) analysis and then by the within-subjects contrasts (WSC) to demonstrate the potential shape of the time or group x time interaction
with significance indicated by the following super/subscripts: * indicates p<0.05 p-level significance, § indicates p<0.10 p-level significance. LSD post hoc analysis is indicated by the following superscripts: ^ represents
p<0.05 difference between groups, † represents p<0.05 difference from baseline value, Ψ represents p<0.05 difference from pre-lift, ◊ represents p<0.05 difference from 60-min post, # represents p<0.05 difference
from 24-hr post. AST Aspartate aminotransferase, ALT Alanine aminotransferase, BUN blood urea nitrogen, CK Creatine kinase, G group p-level, T time p-level, G x T interaction, q quadratic p-level, L linear p-level

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Page 13 of 23

Fig. 4 Markers of protein catabolism. Data expressed as means ± SE and significance indicated by the following super/subscripts: * indicates p < 0.05
p-level significance, § indicates p < 0.10 p-level significance. LSD post hoc analysis is indicated by the following superscripts: ^ represents p < 0.05 difference
between groups, Ψ represents p < 0.05 difference from pre-lift, ◊ represents p < 0.05 difference from 60-min post, # represents p < 0.05 difference from24-h post

counts 24-h and 48-h post-lift in TC compared to P, but
unlike those supplementing with P, TC lymphocyte counts
returned to pre-lift values by the end of the recovery (48-hpost-lift). A significant group effect over time was also reported for WBC (p = 0.020). WBC decreased on average
8 % below pre-lift values during the recovery in P, but only
3 % in TC. Post-hoc analysis demonstrated significant
WBC differences pre-lift, 24-h, and 48-h post-lift between

TC and P that became insignificant once changes were
calculated from pre-lift levels.

Discussion
This is the first study to investigate the effect of freeze
dried Montmorency tart cherry skin powder on acute resistance exercise performance and recovery. It was hypothesized that supplementation with this novel powdered tart

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Page 14 of 23

Fig. 5 Markers of physiological stress and secondary indices of muscle damage. Data expressed as means ± SE and significance indicated by thefollowing super/subscripts: * indicates p < 0.05 p-level significance, § indicates p < 0.10 p-level significance. LSD post hoc analysis is indicated by
the following superscripts: ^ represents p < 0.05 difference between groups, Ψ represents p < 0.05 difference from pre-lift, ◊ represents p < 0.05
difference from 60-min post, # represents p < 0.05 difference from 24-h post

cherry skin supplement surrounding an acute bout ofintense resistance exercise would reduce perceptions ofmuscle soreness, markers of muscle damage, oxidative
stress, and inflammation, thus better maintaining subsequent performance within the first 48-h of recovery. The
results of the present study demonstrate that this poweredtart cherry formulation is effective in promoting decreased
perceptions of muscle soreness following intense resistance
exercise. Tart cherry powder reduced perceptions ofmuscle soreness in the distal vastus medalis and lateralis.
In accordance with decreased perceptions of muscle soreness, tart cherry powder supplementation reduced serum
markers of muscle catabolism and physiological stress overthe 48-h post-lift period compared to placebo. As a resultof the resistance exercise, markers of oxidative stress, lipid
peroxidation, and antioxidant activity did not significantly
change over time as was not affected by differences in supplementation. The inflammatory response deviated from

Variable

Group

Baseline

Pre-Lift

60-min Post

24-hr Post

48-hr Post

Group Meanp-value (GG)

Cortisol (ug/dL)P

28.34±13.07^

23.33±8.80

26.37±7.33

25.08±10.21^

27.68±11.20^

26.16±2.18

G=0.167TC

21.48±7.02

20.49±7.92

26.81±12.08†

19.72±5.56◊

19.74±6.82◊

21.65±2.28

T=0.093§

TL=0.611

21.91±1.75

Ψ◊23.71±1.96

G X T=0.170

G X Tq=0.029*

Time Mean
Testosterone (ng/mL)

Test/Cort Ratio

24.91±2.2226.59±2.06

22.40±1.74

p-value (WSC)P

9.86±3.46

9.37±3.45

9.25±2.92

10.17±3.55

9.94±3.429.72±0.75

G=0.025*TC

7.24±2.22

7.17±2.00

6.97±1.95

7.20±2.00

6.91±1.70

7.10±0.79

T=0.428

Tq=0.427

G X T=0.556

G X TL=0.174

Time Mean

8.55±0.61

8.27±0.60

8.11±0.52

8.68±0.61

8.42±0.57

P

0.037±0.009

0.043±0.014

0.037±0.013

0.045±0.019

0.040±0.018

0.040±0.003

G=0.538TC

0.037±0.015

0.040±0.020

0.031±0.015

0.039±0.013

0.040±0.019

0.037±0.004

T=0.152

TL=0.516

Time Mean

0.037±0.012

0.042±0.017

0.034±0.014

0.042±0.016

0.040±0.018

G X T=0.815

G X Tq=0.196

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Table 6 Anabolic/Catabolic Hormone Response

Individual group and time data expressed as means ± SD, while group effects are presented as means ± SEM. Data represents the stress and sex hormone response at each test session during the 10 day intervention.
MANOVA analysis revealed overall Wilks' Lambda time (p=0.293) and group x time (p=0.458). Univariate ANOVA p-levels from MANOVA analysis are presented for each variable. Univariate ANOVA p-levels are listed
first by the Greenhouse-Geisser (GG) analysis and then by the within-subjects contrasts (WSC) to demonstrate the potential shape of the time or group x time interaction with significance indicated by the following
super/subscripts: * indicates p<0.05 p-level significance, § indicates p<0.10 p-level significance. LSD post hoc analysis is indicated by the following superscripts: ^ represents p<0.05 difference between groups, †represents p<0.05 difference from baseline value, Ψ represents p<0.05 difference from pre-lift, ◊ represents p<0.05 difference from 60-min post, # represents p<0.05 difference from 24-hr post. Test/Cort =Testosterone/Cortisol ratio G group p-level, T time p-level, G x T interaction, q quadratic p-level; L = linear p-level

Page 15 of 23

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Page 16 of 23

Table 7 Markers of Free Radical Production and Oxidative Stress
VariableGroup

Baseline

Pre-Lift

60-min Post

24-hr Post

48-hr Post

Group Meanp-value (GG)

Nitrotyrosine (nM)P

305±267306±311297±289297±330294±305

300±74

G=0.940TC

303±193319±254277±190275±202283±203292±78T=0.280

TL=0.146

Time Mean

304±230312±279288±241287±270289±256Ψ

G X T=0.613

G X TL=0.483

TBARS (uM)

TAS (mM)

SOD (U/mL)

p-value (WSC)P

7.84±2.69

7.27±2.91

8.60±2.67

8.03±3.34

8.43±3.74

8.03±0.81

G=0.913TC

7.74±4.24

8.24±4.03

7.44±3.84

8.19±3.46

7.91±3.35

7.90±0.84

T=0.937

TL=0.539

Time Mean

7.79±3.44

7.73±3.44

8.05±3.26

8.10±3.32

8.18±3.49

G X T=0.569

G X TL=0.649

P

2.90±1.34

2.83±1.77

3.02±1.85

2.95±1.47

2.80±1.81

2.90±0.39
2.72±0.41

TC

2.69±1.17

2.39±1.14

2.99±1.07

2.73±1.36

2.81±1.34

Time Mean

2.80±1.23

2.62±1.48

3.01±1.49

2.84±1.39

2.81±1.57

G=0.752
T=0.323

Tq=0.419

G X T=0.706

G X TL=0.442

P

0.58±0.07

0.59±0.05

0.59±0.09

0.56±0.08

0.58±0.06

0.58±0.02

G=0.668TC

0.55±0.12

0.60±0.09

0.54±0.09

0.57±0.10

0.57±0.11

0.57±0.02

T=0.264

Tq=0.827

0.56±0.10†

0.57±0.09

0.57±0.08

0.58±0.09

G X T=0.335

G X TL=0.784

Time Mean

0.60±0.07

Individual group and time data expressed as means ± SD, while group effects are presented as means ± SEM. Data represents the response to reactive oxygenand nitrogen species production in addition to antioxidant activity at each test session during the 10 day intervention. MANOVA analysis revealed overall Wilks'
Lambda time (p=0.321) and group x time (p=0.756). Univariate ANOVA p-levels from MANOVA analysis are presented for each variable. Univariate ANOVA p-levels
are listed first by the Greenhouse-Geisser (GG) analysis and then by the within-subjects contrasts (WSC) to demonstrate the potential shape of the time or group xtime interaction with significance indicated by the following super/subscripts: * indicates p<0.05 p-level significance, § indicates p<0.10 p-level significance. LSD
post hoc analysis is indicated by the following superscripts: ^ represents p<0.05 difference between groups, † represents p<0.05 difference from baseline value, Ψrepresents p<0.05 difference from pre-lift, ◊ represents p<0.05 difference from 60-min post, # represents p<0.05 difference from 24-hr post
TBARS Thiobarbituric acid reactive substances, TAS Total antioxidant status, SOD superoxide dismutase, G group p-level, T time p-level, G x T interaction, qquadratic p-level, L linear p-level

pre-lift values during the recovery period, but the change
was not different between supplement groups.
The attenuation of quadriceps muscle soreness in conjunction with reduced hemodynamic markers of muscle
catabolism following the resistance training bout indicates that the consumption of tart cherry powder may
have dampened the effects of the secondary muscle
damage response. The initial injury to the muscle microstructure is defined by a mechanical disruption of themyofibrils, particularly in response to high volume andintensity eccentric exercise [9, 44, 56]. The damage tothe muscle microstructure triggers a local inflammatory
response characterized by infiltration of fluid, plasma
proteins, and free radicals that all exacerbate the initialmechanical muscle damage, creating a secondary injury
incidence [9, 44, 57–60]. Decreased perceptions of quadriceps muscle soreness and muscle catabolism indicesreveal that supplementation with powdered tart cherry
may aid in blunting the secondary muscle damage effects compared to placebo.
The apparent beneficial effect of tart cherry powder supplementation on the perception of muscle soreness after aresistance exercise bout is consistent with some of thepreviously published findings. Connolly et al. [20] reported
that consumption of a Montmorency tart cherry juice inhealthy college-aged males significantly reduced pain in
the elbow flexors after a bout of eccentric exercise using avisual analog scale (VAS). Peak muscle pain was achieved

24-h post-exercise in the tart cherry group compared to acontinued increase in pain 48-h post-exercise in theplacebo group. In contrast, Connolly et al. [20] reported
no difference in pressure pain threshold (PPT) scores
between the two experimental groups. Using a strenuous
single leg knee extension protocol in a cohort of welltrained male athletes, Bowtell et al. [4] demonstrated atrend in post-exercise muscle pain reduction via PPT with
Montmorency tart cherry juice supplementation up to 48h post-exercise compared to placebo. The positive resultsin the current study are similar to previous tart cherry
supplementation research findings, showing that powdered tart cherry supplementation significantly attenuatesmuscle soreness perceptions throughout the 48-h post-lift
recovery compared to placebo. Slight differences instudy outcomes may lie in the disparity of muscle painor soreness measures, tart cherry supplements, quantityof musculature involvement, and/or exercise modality.
Measurement of muscle soreness perception in thepresent study utilizing both an algometer and a GRPS was
implemented to help ameliorate the purely subjective
nature of a VAS as the only measure of pain or soreness.
The attenuation in post-lift muscle soreness with powdered tart cherry supplementation may be described byunderlying catabolic mechanisms. Similar to the results ofthe current study, Bowtell et al. [4], following exhaustive
leg extension exercise, demonstrated a faster post-exercise
recovery of knee extensor force coupled with a trend for aLevers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Page 17 of 23

Table 8 Pro-inflammatory Cytokines and Chemokines
VariableGroup

Baseline

Pre-Lift

60-min Post

24-hr Post

48-hr Post

Group Meanp-value (GG)

TNF-α (pg/mL)

P

2.82±1.33

3.30±1.59

3.33±1.88

2.98±1.45

2.85±1.41

3.05±0.36

G=0.212TC

2.12±0.92

2.55±1.15

2.61±1.00

2.44±0.82

2.23±0.94

2.39±0.37

T=0.003*

Tq=0.001*

Time Mean

2.47±0.24

2.92±0.29†

2.97±0.32†

2.71±0.25◊

2.54±0.25Ψ◊G X T=0.873

G X TL=0.649

P

14.92±31.51

18.91±44.08

18.99±44.51

21.33±51.78

21.56±51.82

19.14±9.41

G=0.365TC

6.19±5.69

6.46±5.01

7.12±5.19

7.11±5.98

5.78±6.02

6.53±9.83

T=0.287

Tq=0.136

G X T=0.314

G X TL=0.295

IFN-γ (pg/mL)

IL-1β (pg/mL)

IL-2 (pg/mL)

IL-6 (pg/mL)

IL-8 (pg/mL)

IL-12p70 (pg/mL)#Time Mean

10.56±4.83

12.68±6.70

13.06±6.77

14.22±7.87

13.67±7.88

P

0.77±0.32

0.84±0.35

0.87±0.39

0.83±0.38

0.83±0.35

0.83±0.10TC

0.75±0.35

0.75±0.34

0.82±0.26

0.85±0.30

0.82±0.34

0.80±0.10

Time Mean

0.76±0.07

0.80±0.07†

0.85±0.07†

0.84±0.07

0.82±0.07

p-value (WSC)

G=0.048*
T=0.047*

Tq=0.030*

G X T=0.411

G X Tq=0.488

P

1.86±1.86

2.00±2.18

1.97±2.16

1.91±2.15

1.87±2.09

1.92±0.71

G=0.956TC

1.70±2.96

1.73±2.73

2.06±2.65

2.26±3.13

2.15±3.03

1.98±0.74

T=0.292

TL=0.214Time Mean

1.78±0.51

1.87±0.51

2.02±0.50

2.08±0.56

2.01±0.54

G X T=0.230

G X TL=0.163

P

1.19±0.95

1.34±1.36

1.61±1.60

1.33±1.38

1.26±1.21

1.35±0.37

G=0.724TC

0.87±1.34

1.09±1.31

1.54±1.38

1.20±1.49

1.07±1.43

1.16±0.39

Time Mean

1.03±0.24

1.22±0.28

1.57±0.31†Ψ

1.27±0.30†

1.17±0.28◊#

T=0.020*

Tq=0.023*

G X T=0.761

G X TL=0.512P

4.25±3.99

5.07±5.74

5.04±6.16

5.16±6.41

4.94±6.32

4.89±1.23

G=0.496TC

2.95±1.56

3.45±1.75

4.14±1.29

4.02±1.78

3.74±1.91

3.66±1.28

T=0.042*

Tq=0.018*

Time Mean

3.60±0.64†

4.26±0.90†

4.59±0.95

4.59±1.00

4.34±0.99

G X T=0.697

G X TL=0.736

P

2.27±4.13

3.03±6.51

3.05±6.63

3.01±6.42

2.78±5.68

2.83±1.33

G=0.577TC

1.70±2.67

1.76±2.59

1.68±2.15

1.91±2.76

1.66±2.61

1.74±1.39

T=0.279

Tq=0.264

Time Mean

1.99±0.73

2.39±1.05

2.37±1.05

2.46±1.05

2.22±0.94

G X T=0.377

G X TL=0.351

Individual group and time data expressed as means ± SD, while group effects are presented as means ± SEM. Data represents the pro-inflammatory response ateach test session during the 10 day intervention. MANOVA analysis revealed overall Wilks' Lambda time (p<0.001) and group x time (p=0.302). Univariate ANOVA
p-levels from MANOVA analysis are presented for each variable. Univariate ANOVA p-levels are listed first by the Greenhouse-Geisser (GG) analysis and then by
the within-subjects contrasts (WSC) to demonstrate the potential shape of the time or group x time interaction with significance indicated by the following super/
subscripts: * indicates p<0.05 p-level significance, § indicates p<0.10 p-level significance. LSD post hoc analysis is indicated by the following superscripts: ^represents p<0.05 difference between groups, † represents p<0.05 difference from baseline value, Ψ represents p<0.05 difference from pre-lift, ◊ represents
p<0.05 difference from 60-min post, # represents p<0.05 difference from 24-hr post
TNF-α tumor necrosis factor alpha, IFN-γ interferon gamma, IL interleukin, G group p-level, T time p-level, G x T interaction, q quadratic p-level, L linear p-levelsmaller CK percent change from pre-exercise levels up to
48-h post-exercise with tart cherry supplementation compared to placebo [43]. Following a treadmill incremental exercise protocol with thoroughbred horses, Ducharme et al.
[61] reported an attenuation trending toward significancein CK levels during exercise recovery when supplementing
with a tart cherry juice blend compared to placebo. Similar
to the post-lift attenuations of ALT (60-min and 48-h post)
and AST (48-h post) as secondary markers of muscle damage in the current study, Ducharme et al. [61] reported that
AST was significantly mitigated during and following an
incremental exercise protocol in the tart cherry-treatedhorses compared to placebo [43]. In an endurance-based
crossover study examining the effects of acute ibuprofen(8400 mg) ingestion surrounding a 45-min downhill treadmill run, Donnelly et al. [62] reported an increase in serum
CK, AST, and lactate dehydrogenase post-run with maximal activity of CK and AST occurring at 24-h post-run,

while lactate dehydrogenase, creatinine, and urea peaked
6-h post-run. Donnelly et al. [62] revealed that serum CK
and urea levels were significantly higher after ibuprofen
supplementation compared to placebo throughout the
post-run recovery. Analyzing hemodynamic clinical chemistry makers before, 4-h, and 24-h post-Boston marathon,Kratz et al. [63] reported significant increases in total CK,
AST, ALT, total protein, uric acid, and creatinine 4-h postrace and confirmed that CK, creatinine, uric acid, ALT,
and AST remained significantly elevated over pre-racevalues 24-h post-race. The attenuation of these markers in
the current study, demonstrates beneficial powdered tart
cherry supplementation effects on the post-resistance
exercise catabolic, muscle damage, and stress response.
Acute, intensive resistance exercise (>60 % 1-RM) has
been reported to significantly increase muscular mechanical trauma as the initial phase of injury [4, 64]
coupled with a secondary inflammatory injury phase that

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Page 18 of 23

Table 9 Anti-inflammatory Cytokines
VariableGroup

Baseline

Pre-Lift

60-min Post

24-hr Post

48-hr Post

Group Meanp-value (GG)

IL-4 (pg/mL)

P

6.03±3.18

7.86±5.06

8.26±5.22

7.44±4.95

7.83±5.10

7.48±1.05

G=0.273TC

4.16±1.91

5.45±2.04

6.38±2.49

6.36±2.45

6.54±3.01

5.78±1.09

T=0.001*

Tq=0.001*

Time Mean

5.10±0.55

6.65±0.82†

7.32±0.87†

6.90±0.83†◊

7.19±0.88†

G X T=0.421

G X TL=0.405

IL-5 (pg/mL)

IL-7 (pg/mL)

IL-10 (pg/mL)

IL-13 (pg/mL)

p-value (WSC)P

0.69±0.37

0.70±0.41

0.70±0.46

0.64±0.37

0.65±0.40

0.68±0.11

G=0.749TC

0.75±0.43

0.72±0.40

0.71±0.37

0.77±0.43

0.70±0.42

0.73±0.12

T=0.716

TL=0.325

Time Mean

0.72±0.08

0.71±0.09

0.70±0.09

0.71±0.08

0.68±0.09

G X T=0.438

G X TL=0.582

P

4.73±2.19

5.41±2.49

5.94±3.71

5.52±2.75

5.46±2.57

5.41±0.63

G=0.414TC

4.15±1.86

4.45±2.00

5.31±2.18

4.91±1.61

4.47±1.54

4.66±0.65

Time Mean

4.44±0.43

4.93±0.48†

5.62±0.64†

5.21±0.48†

4.97±0.45†

T=0.041*

Tq=0.033*

G X T=0.843

G X TL=0.757

P

4.52±3.85

3.69±1.82

4.21±1.92

3.48±1.70

3.45±1.74

3.87±0.56

G=0.869TC

2.77±1.74

3.49±2.03

6.02±5.70

3.33±2.54

3.07±2.54

3.74±0.59

T=0.103§

Tq=0.157

G X T=0.182

G X Tq=0.120

ΨTime Mean

3.65±0.63

3.59±0.40

5.12±0.87

3.41±0.45

3.26±0.45P

3.30±2.89

3.33±3.30

3.40±3.48

3.24±3.64

3.27±3.56

3.31±0.78

G=0.449TC

1.96±1.74

2.26±1.66

2.75±2.09

2.78±2.18

2.45±2.34

2.44±0.81

T=0.393

Tq=0.055§

Time Mean

2.63±0.50

2.80±0.55

3.08±0.61

3.01±0.63

2.86±0.64

G X T=0.402

G X Tq=0.133

Individual group and time data expressed as means ± SD, while group effects are presented as means ± SEM. Data represents the anti-inflammatory response ateach test session during the 10 day intervention. MANOVA analysis revealed overall Wilks' Lambda time (p<0.001) and group x time (p=0.447). Univariate ANOVA
p-levels from MANOVA analysis are presented for each variable. Univariate ANOVA p-levels are listed first by the Greenhouse-Geisser (GG) analysis and then by
the within-subjects contrasts (WSC) to demonstrate the potential shape of the time or group x time interaction with significance indicated by the following super/
subscripts: * indicates p<0.05 p-level significance, § indicates p<0.10 p-level significance. LSD post hoc analysis is indicated by the following superscripts: ^represents p<0.05 difference between groups, † represents p<0.05 difference from baseline value, Ψ represents p<0.05 difference from pre-lift, ◊ represents
p<0.05 difference from 60-min post, # represents p<0.05 difference from 24-hr post
IL interleukin, G group p-level, T time p-level, G x T interaction, q quadratic p-level, L linear p-leveloccurs as a result of plasma protein and inflammatory cell
infiltration of the damaged tissue [4, 9, 57]. Howatson etal. [44] reported significantly lower IL-6 concentrations
after running a marathon that coincided with quicker
recovery of knee extensor maximal strength following the
marathon in Montmorency tart cherry juice supplemented
subjects compared to placebo [44]. As an inflammatory
marker, IL-6 was significantly attenuated immediately
post-race with tart cherry consumption compared to placebo [43, 44]. In a strength-based supplement study withresistance trained subjects, Bowtell et al. [4] was also notable to detect any significant effects on IL-6 as a marker
of inflammation. Further, Trombold et al. [65] reported
similar non-significant inflammatory (IL-6) findings in astudy involving non-resistance trained subjects performing repeated bouts of eccentric elbow flexor muscle contractions with consumption of pomegranate-derived
ellagitannin [4]. Supplementation in the current study did
not affect the inflammatory response to muscular trauma
as markers peaked 60-min post-lift with no response differences between supplementation groups. Attenuation ofmuscle soreness perceptions and markers of muscle damage are not likely attributed to changes in inflammation
following a single resistance exercise bout. Differences inresults between studies are likely attributed to variation in

intensity, duration, and modality of the aforementioned
exercise protocols.
The increase in inflammatory-related cytokines andchemokines such as TNF-α, IL-1β, IL-6, and IL-8 during
and immediately following exercise has been shown inprevious research [66–68] and closely resembles the inflammatory response in the current study. As describedin previous literature, the increase in serum IL-6 withinboth groups 60-min post-lift in the current study canlikely be attributed to the large release of muscle-derived
IL-6 as a result of the contracting muscle fibers [66, 69].
The influx of muscle-derived IL-6 within the systemic
circulation may be one of the triggers for subsequentcortisol release in response to exercise-induced stress[66, 70]. Glucocorticoids, specifically cortisol, and antiinflammatory cytokines such as IL-4, IL-10, and IL-13
are released during strenuous exercise and typicallydemonstrate an immunosuppressive influence to help
modulate the immune response balance [71]. However,extended immunodepression due to elevated plasma
cortisol during the post-exercise period, particularly following long bouts of intense training in the fasted state[66], may hinder recovery and subsequent performance.
Previous research conducted by McAnulty et al. [72] intrained endurance athletes reported a significant cortisol

Variable

Group

Baseline

Pre-Lift

60-min Post

24-hr Post

48-hr Post

Group Meanp-value (GG)

LYMPH (K/uL)

P

1.93±0.54

2.21±0.69

1.45±0.43†Ψ

1.86±0.71^Ψ◊

1.78±0.48^Ψ◊

1.85±0.20

G=0.311TC

2.09±0.86

2.36±1.05†

1.49±0.58†Ψ

2.62±1.51†Ψ◊2.15±0.78◊#

2.14±0.21

T<0.001*

Tq=0.136

Time Mean

2.01±0.15†

2.28±0.18

1.47±0.11†Ψ

G X T=0.143

G X TL=0.015*

P

5.88±1.23

6.61±2.21^

6.38±1.45

6.16±1.62^

5.75±1.61^

6.16±0.40

G=0.337TC

5.54±1.25

7.18±2.03†

6.98±2.04†

7.40±2.17†

6.55±1.23†#

6.73±0.42

T=0.004*

Tq=0.004*

Time Mean

5.71±0.26†

6.90±0.44

6.68±0.37††

6.15±0.30Ψ

G X T=0.207

G X TL=0.020*P

0.43±0.12

0.64±0.19

0.43±0.14

0.53±0.13

0.48±0.13

0.50±0.03

G=0.866

TC

0.42±0.10

0.61±0.24

0.38±0.10

0.60±0.19

0.55±0.16

0.51±0.03

T<0.001*

TL=0.100§

Time Mean

0.43±0.02

0.63±0.05†

0.41±0.03Ψ

0.56±0.03†◊

0.51±0.03†Ψ◊

G X T=0.324

G X TL=0.062§

P

3.50±0.97

3.76±1.47

4.51±1.20

3.78±1.33

3.48±1.32

3.80±0.33

G=0.577TC

3.04±0.48

4.22±1.77

5.11±2.35

4.17±1.61

3.84±0.98

4.08±0.35

T<0.001*

Tq=0.002*

Time Mean

3.27±0.16

3.99±0.34†

4.81±0.38†Ψ

3.97±0.31†◊

3.66±0.03◊

G X T=0.332

G X TL=0.119

WBC (K/uL)

MID (K/uL)

GRAN (K/uL)

GM-CSF (pg/mL)

2.24±0.24

6.78±0.40◊

1.97±0.13

Ψ◊p-value (WSC)P

8.12±8.23

8.18±8.33

7.87±7.38

7.54±7.67

7.39±7.49

7.82±2.71

G=0.512TC

10.37±11.40

10.51±10.83

10.52±10.48

10.67±11.19

10.09±10.89

10.43±2.83

T=0.446

TL=0.277

Time Mean

9.24±2.06

9.34±2.00

9.20±1.88

9.10±1.99

8.74±1.93

G X T=0.624

G X TL=0.450

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Table 10 Markers of Immune-Related Complete Blood Counts

Individual group and time data expressed as means ± SD, while group effects are presented as means ± SEM. Data represents immune cellular response markers at each test session during the 10 day intervention.
MANOVA analysis revealed overall Wilks' Lambda time (p<0.001) and group x time (p=0.684). Univariate ANOVA p-levels from MANOVA analysis are presented for each variable. Univariate ANOVA p-levels are listed
first by the Greenhouse-Geisser (GG) analysis and then by the within-subjects contrasts (WSC) to demonstrate the potential shape of the time or group x time interaction with significance indicated by the following
super/subscripts: * indicates p<0.05 p-level significance, § indicates p<0.10 p-level significance. LSD post hoc analysis is indicated by the following superscripts: ^ represents p<0.05 difference between groups, †represents p<0.05 difference from baseline value, Ψ represents p<0.05 difference from pre-lift, ◊ represents p<0.05 difference from 60-min post, # represents p<0.05 difference from 24-hr post
LYMPH lymphocytes, WBC white blood cell, MID mid-range absolute count, GRAN granulocyte absolute count, GM-CSF granulocyte-macrophage colony-stimulating factor, G group p-level, T time p-level, G x Tinteraction, q quadratic p-level, L linear p-level

Page 19 of 23

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

increase over time, but no significant differences in immediate and 1.5-h post-race cortisol levels with 2-month vitamin E supplementation compared to placebo. Comparing
14-d supplementation of N-acetyl-cysteine (NAC), epigallocatechin gallate (EGCG), or a placebo in active males
surrounding a single bout of eccentric knee-extensor exercise, Kerksick et al. [73] found a significant decrease inserum cortisol levels 6-h post-exercise, but no differences
between groups up to 72-h post-exercise despite a significant blunting of muscle soreness perception 24-h post exercise in the two supplement groups compared to placebo.
The current study demonstrated an increase in cortisol
levels 60-min post-lift compared to pre-lift in both groups.
However, unlike the cortisol and muscle soreness perception results reported by Kerksick et al. [73], the current
study demonstrated significantly elevated cortisol levels
and muscle soreness in the placebo group compared tothe powdered tart cherry group 24-h and 48-h post-lift.
The placebo group elevations in serum cortisol levelsin the latter recovery period may be linked to differencesin the immune response and the degree of secondary
muscle injury. Previous research by McCarthy et al. [74]and Robson et al. [75] demonstrate that in response torepetitive muscular contraction during and immediately
after acute, intense exercise, there is a large increase incirculating neutrophils (GRAN) and lymphocytes. According to data collected by Robson et al. [75], plasma
neutrophil levels tend to peak 2–3 hours post-exercise,but remain elevated above pre-exercise levels for up to
10-h post-exercise due to elevations in plasma cortisol(e.g. perception of stress) that facilitate the release ofneutrophils from the bone marrow. The current study
demonstrated a plasma neutrophil (GRAN) peak 60-min
post-lift, but effects of supplementation could not be
determined as levels returned to pre-lift concentrations
by 24-h. Unlike the neutrophil response to acute exercise, lymphocytes respond in a biphasic pattern, whereplasma levels are significantly elevated during and immediately post-exercise followed by a drop below preexercise levels during the initial recovery period due to aefflux from the blood circulation, and a slow return tobaseline levels as equilibrium is reached again [71, 75].
Robson et al. [75] demonstrated that serum cortisol
levels influence both the influx of neutrophils and effluxof lymphocytes from systemic circulation. The results ofthe current study demonstrated that lymphocyte levelsin the placebo group never returned to pre-lift levels
during the 48-h recovery. This may be linked to the elevated plasma cortisol levels in the placebo group representing an increased physiological perception of exercise
stress 48-h post-lift compared to pre-lift and powderedtart cherry group levels. As previously mentioned, thesecondary injury phase occurs as a result of significant
plasma protein and inflammatory cell influx within the

Page 20 of 23

damaged tissue [4, 9, 57]. Powdered tart cherry supplementation may have aided in reducing secondary muscular injury through an initial dampening of the immune
response paired with the attenuated cortisol response
later in exercise recovery. However, immune cell countchanges in response to supplementation and the resistance exercise stimulus are speculative due to the lack of
cellular count data corresponding to typical patterns of
immune cell responses during and immediately following the barbell back squat challenge.
The increase in antioxidant bioavailability from tart
cherries containing high levels of flavonoids and anthocyanins [76, 77] has been hypothesized to be one of themain benefits of tart cherry supplementation. Antioxidant bioavailability is important in maintaining adequate
redox balance to support the endogenous antioxidant
systems following excessive ROS-producing strenuous
exercise. Ducharme et al. [61] investigated the effects ofMontmorency tart cherry juice supplementation on oxidative stress in thoroughbred horses following a stepwise
incremental treadmill running protocol [43]. Overall indications of oxidative stress as a result of exercise werereported through significant elevations in lipid hydroperoxidation (TBARS) [61], but similar to the current study,
no differences between groups were shown. In an endurance study examining the effects of tart cherry juice supplementation on oxidative stress following a marathon
run, Howatson et al. [44] demonstrated significantly lower
TBARS levels in the tart cherry supplemented groupversus placebo 48-h post-marathon. As a highly reactiveoxide metabolite of nitric oxide, peroxynitrate-bound tyrosine residues forming nitrotyrosine (NT) [78] were measured by Sureda et al. [79] following supplementation ofvitamin C + E surrounding a half-marathon. Suerda et al.
[79] reported a significant increase in both NT immediately post-race and 3-h post-race in the placebo groupcompared to the vitamin C + E supplemented group, indicating that antioxidant supplementation may have anattenuating effect on the oxidation of nitrogen-containing
compounds surrounding endurance exercise. Contrarily,both Bowtell et al. [4] and the current study found no difference between supplementation groups with regard tooxidation of nitric oxide (NT) in the post-strength exercise
period. A difference in outcome may be attributable to thedifference in exercise modality and thus the minimal reliance on aerobic metabolic demands during resistance
exercise. Further, evidence in the literature utilizing lipid
peroxidation (TBARS) analyses have presented a potential
lack of oxidative damage detection specificity in humanstudies that may also explain the variability in results
between the current and previous studies [11, 43, 80, 81].
In a study analyzing the recovery from a marathon
run, Howatson et al. [44] analyzed plasma TAS, as ameasure of antioxidant or antiradical activity [82], andLevers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

found that TAS fell below baseline 48-h post-marathonin the placebo group [43]. Unlike the tart cherry group,
the placebo group failed to maintain TAS or redox balance following the endurance exercise, demonstrating
possible antioxidant effectiveness on excessive ROS production with bouts of endurance exercise [43]. Childs
et al. [83] analyzing TAS following arm eccentric exerciseinduced injury demonstrated significantly greater TAS
levels with vitamin C + NAC supplementation comparedto placebo 48-h through 7-d post-exercise in healthy,
untrained males. A resistance training-related study conducted by Lafay et al. [84] investigating the effects of apolyphenol-rich grape extract on repeated jumping force
capacity, found a significant jumping force capacity
improvement in the grape extract group over placebo.
However, similar to the current study, Lafay et al. [84]found no significant difference in SOD activity between
the groups. The current resistance training study also did
not demonstrate any significant change in TAS activity
across time or between groups possibly attributed to differences in study population and the low metabolic costassociated with resistance/eccentric exercise versus endurance exercise.
The strengths of this particular study revolve around theanalysis of a large cohort of muscle soreness, performance,
muscle damage, inflammatory, and oxidative damage measures to contribute a comprehensive analysis to the existing
body of published literature. This type of comprehensive
hemodynamic analysis has not been conducted in a tart
cherry supplement study paired with resistance exercise.
Further, the current study employed one of the largest subject cohorts among recent studies examining polyphenolic,vitamin, or NSAID supplementation effects on performance and related hemodynamic markers. The utilization ofthis supplement within the free-living resistance trained
population demonstrated its effectiveness under normative
training, diet, and performance conditions. The ease associated with consuming an encapsulated powdered supplement versus a potentially inconvenient or unpalatable juice
most likely enhanced subject compliance with supplementation. Potential limitations and weakness of the current
study should also be considered. The placebo-control
matched design of this study was effective in equalizing
study subject exposure to the resistance protocol irrespective of supplement group, however compared to across-over design, there might have been some variability associated with subject pairing. Differences in resistance training experience and overall state of training
beyond the study inclusion/exclusion criteria may also
have been a source of variability in study cohort recruitment. Due to the large number of hemodynamic markers
measured in this study, the five selected time points ofblood draws over the course of the experimental period
may have not captured the entire pharmacokinetic profile

Page 21 of 23

for each marker. Despite controlling 48-h training activity
and NSAID intake with a 10-h fast before each session,
nutritional intake and hydration status of each subject was
not controlled nor tested. Differences in nutrition and hydration among study subjects could have been a potential
source of hemodynamic marker or performance measure
variability in a cohort of this size. Further, the 48-hour
NSAID ingestion limitation may not have been a sufficientwashout time for all NSAID-type medications considering
the large range in reported half-life of common NSAIDs(1.6 to 30-h). The major overriding strengths of this study
are that this is the first practical study to be conductedsurrounding an acute bout of strength exercise incorporating significant muscle mass recruitment and it is the firststudy to be conducted utilizing a powdered form of cherries rather than a juice or concentrate.

Conclusions
The current study demonstrated that consumption of aMontmorency powdered tart cherry supplement 7-d
before, the day of, and 2-d after completing a single boutof high volume, high-intensity resistance exercise, appears to be an effective dietary supplement in reducing
muscle soreness across the most biomechanically loaded
region of the quadriceps near the distal patellar attachment and markers of muscle catabolism in resistancetrained individuals. Short-term supplementation with powdered tart cherry surrounding a single bout of resistancetraining did not demonstrate any definitive effect onmarkers of oxidative damage or inflammation. Due to theinconclusive oxidative damage and inflammatory evidence,mechanisms of short-term powdered tart cherry and otherrelated phytochemical-containing nutritional supplements
surrounding bouts of high intensity, anaerobic and resistance exercise need to be further investigated. Additional
examination of powdered tart cherry supplementation withother forms of exercise that are known to promote a more
pronounced effect on inflammation and oxidative stress(e.g. endurance exercise) is also needed. However, the initialeffectiveness in reducing perceptions of muscle sorenessand markers of muscle catabolism in resistance-trained
men demonstrates that powdered tart cherry supplementation provides similar benefits as previously studied tart
cherry juices or concentrates following acute bouts of lower
body strength-based exercise.
Competing interests
This study was funded by Anderson Global Group, LLC (Irvine, CA, USA) and
Shoreline Fruit, LLC (Traverse City, MI, USA) through an unrestricted research
grant to Texas A&M University. All researchers involved independently collected,
analyzed, and interpreted the results from this study and have no financial
interests concerning the outcome of this investigation. RBK serves as auniversity approved scientific consultant for Anderson Global Group, LLC andhas served as a scientific and legal consultant related to exercise and nutrition.
The results from this study do not constitute endorsement by the authors and/or the institution concerning the nutrients investigated.

Levers et al. Journal of the International Society of Sports Nutrition (2015) 12:41

Authors’ contributions
KL assisted in designing the study, writing the grant, study coordination,
data collection, data analysis, and manuscript preparation. RD, CG, AO, SS,
and NB assisted in data collection and sample analysis. EG assisted with datacollection, sample analysis, and reviewed dietary inventories as the Registered
Dietician on staff. CR is the director of the Exercise & Sport Nutrition Lab. MG
assisted in research design and consultation. SMT, SR, and SC assisted in study
consultation. RBK (corresponding author) assisted in the design of the study,
data analysis, manuscript preparation, obtained the grant, and served as thestudy PI. All authors read and approved the final manuscript.
Acknowledgements
We would like to thank the subjects that participated in this study as well as
all of the students and laboratory assistants in the Exercise & Sport Nutrition
Laboratory at Texas A&M University who assisted with data collection. We
would also like to thank Mike Vaughn and Katherine Kelly for their valuableassistance in sample analysis.
Author details
1
Department of Health and Kinesiology, Exercise and Sport Nutrition
Laboratory, Texas A&M University, College Station, TX 77843-4243, USA.
2
United States Military-Baylor University Graduate Program in Nutrition,
AMEDD Center and School, Fort Sam Houston, United States Military, San
Antonio, TX 78234, USA. 3Department of Nutrition and Food Science,
Institute for Obesity Research and Program Evaluation, Texas A&M University,
College Station, TX 77843-4243, USA. 4Department of Health and Kinesiology,
Human Countermeasures Laboratory, Texas A&M University, College Station,
TX 77843-4243, USA. 5Department of Health and Kinesiology, Applied
Exercise Science Laboratory, Texas A&M University, College Station, TX
77843-4243, USA.
Received: 3 August 2015 Accepted: 3 November 2015

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