Effects of protein–carbohydrate supplementation on immunity and resistance training outcomes: a double-blind, randomized, controlled clinical trial

Abstract

PurposeTo examine the impact of ingesting hydrolyzed beef protein, whey protein, and carbohydrate on resistance training outcomes, body composition, muscle thickness, blood indices of health and salivary human neutrophil peptides (HNP1-3), as reference of humoral immunity followed an 8-week resistance training program in college athletes. Methods
Twenty-seven recreationally physically active males and females (n = 9 per treatment) were randomly assigned to one of the three groups: hydrolyzed beef protein, whey protein, or non-protein isoenergetic carbohydrate. Treatment consisted of ingesting 20 g of supplement, mixed with orange juice, once a day immediately post-workout or before breakfast on non-training days. Measurements were performed pre- and post-intervention on total load (kg) lifted at the first and last workout, body composition (via plethysmography) vastus medialis thickness (mm) (via ultrasonography), and blood indices of health. Salivary HNP1-3 were determined before and after performing the first and last workout. ResultsSalivary concentration and secretion rates of the HNP1-3 decreased in the beef condition only from pre-first-workout (1.90 ± 0.83 μg/mL; 2.95 ± 2.83 μg/min, respectively) to pre-last-workout (0.92 ± 0.63 μg/mL, p = 0.025, d = 1.03; 0.76 ± 0.74 μg/min, p = 0.049, d = 0.95), and post-last-workout (0.95 ± 0.60 μg/mL, p = 0.032, d = 1.00; 0.59 ± 0.52 μg/min, p = 0.027, d = 1.02). No other significant differences between groups were observed. Conclusions
Supplementation with a carbohydrate–protein beverage may support resistance training outcomes in a comparable way as the ingestion of only carbohydrate. Furthermore, the ingestion of 20 g of hydrolyzed beef protein resulted in a decreased level and secretion rates of the HNP1-3 from baseline with no negative effect on blood indices of health.

Full text

1 3
Eur J Appl Physiol (2017) 117:267–277
DOI 10.1007/s00421-016-3520-x
ORIGINAL ARTICLE
Effects of protein–carbohydrate supplementation on immunity
and resistance training outcomes: a double‑blind, randomized,
controlled clinical trial
Fernando Naclerio1 · Eneko Larumbe‑Zabala2 · Nadia Ashrafi1 · Marco Seijo1 ·
Birthe Nielsen1 · Judith Allgrove3 · Conrad P. Earnest4
Received: 19 July 2016 / Accepted: 19 December 2016 / Published online: 27 December 2016
© The Author(s) 2016. This article is published with open access at Springerlink.com
indices of health. Salivary HNP1-3 were determined before
and after performing the first and last workout.
Results Salivary concentration and secretion rates of the
HNP1-3 decreased in the beef condition only from pre-
first-workout (1.90 ± 0.83 μg/mL; 2.95 ± 2.83 μg/min,
respectively) to pre-last-workout (0.92 ± 0.63 μg/mL,
p = 0.025, d = 1.03; 0.76 ± 0.74 μg/min, p = 0.049,
d = 0.95), and post-last-workout (0.95 ± 0.60 μg/mL,
p = 0.032, d = 1.00; 0.59 ± 0.52 μg/min, p = 0.027,
d = 1.02). No other significant differences between groups
were observed.
Conclusions Supplementation with a carbohydrate–pro-
tein beverage may support resistance training outcomes in
a comparable way as the ingestion of only carbohydrate.
Furthermore, the ingestion of 20 g of hydrolyzed beef pro-
tein resulted in a decreased level and secretion rates of the
HNP1-3 from baseline with no negative effect on blood
indices of health.
Keywords Immune status · Strength performance · Body
composition · Muscle thickness · Blood indices of health
Abbreviations
AST/GOT Alanine transaminase
AMP Antimicrobial peptides
ALT/GPT Aspartate transaminase
BM Body mass
CHO Carbohydrate
CK Creatine kinase
DHEA Dehydroepiandrosterone
η2
G
Generalized eta squared
HDL High density lipoprotein
HNP1-3 Human neutrophil peptides
LDL Low density lipoprotein
ANOVA One-way analysis of variance
Abstract
Purpose To examine the impact of ingesting hydrolyzed
beef protein, whey protein, and carbohydrate on resistance
training outcomes, body composition, muscle thickness,
blood indices of health and salivary human neutrophil pep-
tides (HNP1-3), as reference of humoral immunity followed
an 8-week resistance training program in college athletes.
Methods Twenty-seven recreationally physically active males
and females (n = 9 per treatment) were randomly assigned
to one of the three groups: hydrolyzed beef protein, whey
protein, or non-protein isoenergetic carbohydrate. Treatment
consisted of ingesting 20 g of supplement, mixed with orange
juice, once a day immediately post-workout or before break-
fast on non-training days. Measurements were performed pre-
and post-intervention on total load (kg) lifted at the first and
last workout, body composition (via plethysmography) vastus
medialis thickness (mm) (via ultrasonography), and blood
Communicated by William J. Kraemer.
Electronic supplementary material The online version of this
article (doi:10.1007/s00421-016-3520-x) contains supplementary
material, which is available to authorized users.
* Fernando Naclerio
f.j.naclerio@gre.ac.uk
1 Department of Life and Sports Sciences, University
of Greenwich, Chatham Maritime, UK
2 Clinical Research Institute, Texas Tech University Health
Sciences Center, Lubbock, TX, USA
3 Faculty of Science Engineering and Computing, Kingston
University, London, UK
4 Exercise Science and Nutrition Laboratory, Texas A&M
University, College Station, TX, USA
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

268 Eur J Appl Physiol (2017) 117:267–277
1 3
Introduction
Heavy exercise can lead to significant transient perturba-
tions in immune functions (Walsh et al. 2011b). In ath-
letes, the so-called immunosuppression has been associ-
ated with a weakened immune state lasting for up to 72 h
after performing a strenuous exercise session (Nieman and
Bishop 2006). Consequently, different nutritional strate-
gies, including the combination of carbohydrates with
high-quality protein sources, ingested throughout the day
(Jones et al. 2015) or during and after workouts or com-
petitions (Naclerio et al. 2015), have been proposed as an
effective nutritional countermeasure to exercise-induced
immune dysfunction with no detrimental effects on indices
of health (e.g., blood chemistry measures) (Antonio et al.
2016). Furthermore, adding protein to augment resistance
training outcomes is supported by their capacity to rap-
idly increase amino acid availability to the working mus-
cles (Wilkinson et al. 2007). Specifically, the post-workout
consumption of high-quality proteins would shift the bal-
ance in favor of muscle anabolism (Rennie et al. 2004) and,
as a consequence, acts to maximize recovery and training
effects between workouts (Morton et al. 2015).
Whey and beef protein extracts are high-quality pro-
tein sources with a similar amino acid composition to that
found in the skeletal muscles (Chernoff 2004; Cruzat et al.
2014). Either whey and beef protein sources include sulfur-
containing amino acids, such as cysteine and methionine or
taurine that have been associated with an efficient immune
status (Reidy and Rasmussen 2016). As a consequence, it
could be possible to hypothesize that the ingestion of high-
quality protein extracts via the provision of an amino acid
would support the immune response in individuals engaged
in exhaustive and intense exercise programs.
Defensins, including alpha-defensins, are antimicro-
bial peptides (AMP) contributing to mucosal host defense
providing a broad spectrum of antibacterial and antifungal
activity (Wang 2014). The alpha-defensins also known as
human neutrophil peptides (mainly HNP1-3) are predomi-
nantly found in neutrophils. Within the last 15 years, sev-
eral investigations have analyzed the response of these
salivary markers of humoral immunology in various oral
and systemic diseases (Yount and Yeaman 2012). Although
some studies have investigated the impact of exercise on
humoral immunity (Gleeson 2000), to the best of authors
knowledge only two studies have analyzed the HNP1-3
response to exercise (Davison et al. 2009; Gillum et al.
2015) and neither of which have investigated the impact of
combining nutritional supplements on the long-term adap-
tation of humoral immunity. Davison et al. (2009) reported
an acute increase in the absolute concentration of the sali-
vary AMP (HNP1-3 and LL-37) in participants having
performed a 2.5 h cycling intervention at 60% of VO2max
while Gillum et al. (2015) observed an acute increase in
the level of four AMP (LL-37, HNP1-3, LL-37, L Lacto-
ferrin, and Lysozyme) in participants after 45 min of run-
ning at 75% VO2peak. The post-exercise increase of salivary
AMP may to some extent be related to an exercise-induced
muscle inflammatory response (Davison et al. 2009). Extra-
neous dynamic exercise may induce airway inflammation
and damage to airway epithelial cell (Davison et al. 2009).
Thus, interventions aimed to analyze long-term adapta-
tion on the humoral immunity without side effects on the
general health would be of relevant importance for ath-
letes. The aim of the current investigation was to compare
the effectiveness of combining an 8-week resistance train-
ing program with a commercially available beef hydro-
lyzed protein powder product, whey isolate or a non-pro-
tein, carbohydrate only supplement on resistance training
outcomes. The primary outcome for the study was sali-
vary alpha-defensins (HNP1-3) and secondary outcomes
included performance, body composition, muscle thick-
ness, and various blood indices of health.
Methods
Participants
Forty-two recreationally active college participants (24
male and 18 females) met the requirements to participate
in this study. Key criteria for inclusion were: (a) 18–40 year
of age, (b) regular recreationally training for at least 2 years
with a minimum of 1 month performing resistance train-
ing, (c) free from musculoskeletal limitations, (d) agree
not to ingest any other nutritional supplements or non-
prescription drugs or medication that might affect the
immune system or muscle growth as well as the ability to
train intensely during the study, and (e) fluent in English.
Key criteria used for exclusion were: (a) history of various
metabolic conditions and/or diseases; (b) use of a variety of
medications, including but not limited to those with andro-
genic and/or anabolic effects and/or nutritional supple-
ments known to improve strength and/or muscle mass such
as creatine, essential amino acid, whey protein, glutamine,
dehydroepiandrosterone (DHEA) within 8 weeks prior to
the beginning of the study; (d) current use of tobacco prod-
ucts; and/or in the case of female participants ingesting
oral contraceptives; and (e) the presence of any orthopedic
limitations or injuries. Compliance was confirmed verbally
with participants upon arrival to the laboratory.
All participants were informed of the potential risks
of the intervention before agreeing to comply with the
intervention protocol and signed an informed consent.
All experimental procedures were conducted in accord-
ance with the Declaration of Helsinki, and approved by
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

269Eur J Appl Physiol (2017) 117:267–277
1 3
the University ethics committee. As summarized in Fig. 1,
after assessing for eligibility, 27 of the 42 recruited par-
ticipants completed all aspects of the study. The study
was conducted during the spring and summer of 2015 at
the Centre for Science and Medicine in Sport and Exercise
University of Greenwich Medway Campus, Kent (UK).
Trial Registration: ClinicalTrials.gov, U.S. National Insti-
tutes of Health. (Identifier: NCT02425020) on 22nd April
2015.
Experimental design
This study was a randomized controlled trial with three
parallel groups, following a double blind between-partic-
ipant design. Participants were randomly allocated into
three treatment groups: beef protein (n = 14); whey pro-
tein (n = 14) or carbohydrate only (CHO, n = 14). Before
(test 1) and after (test 2) an 8-week intervention period,
measurements of performance, body composition and mus-
cular thickness, total cholesterol, low density lipoprotein
(LDL) and high density lipoprotein (HDL) cholesterol,
triglycerides, glucose, urea, uric acid, creatine kinase (CK),
creatinine, alanine transaminase (AST/GOT), aspartate
transaminase (ALT/GPT) and HNP1-3 salivary immune-
peptides were assessed. Additionally, saliva was collected
before and after the first and the last workout of the 8-week
intervention protocol.
After inclusion and before the baseline assessment, par-
ticipants performed six sessions of familiarization, aimed
to minimize any potential learning effects of the training
procedures. Following the pre-intervention assessments,
participants were matched by gender and resistance train-
ing background. Assignment of participants to treatments
was performed by block randomization, using a block
size of three, and in a double-blind fashion. Initial groups
characteristics were as follows: beef: age 25.6 ± 5.7 years,
height 1.72 ± 0.09 m, body mass 74.2 ± 17.3 kg; whey:
age 25.9 ± 5.9 years, height 1.71 ± 0.10 m, body mass
70.2 ± 11.3 kg; CHO: age 24.9 ± 8.1 years, height
1.70 ± 0.09 m, body mass 70.4 ± 15.3 kg. No significant
differences were observed between treatments at inclusion
in sample characteristics.
Fig. 1 Flow diagram of partici-
pants throughout the course of
the study
Assessed for eligibility (n=54)
Excluded (n=12)
Not meeting inclusion criteria (n=10)
Declined to participate (n=0)
Other reasons (n=2)
Analysed (n= 9)
Excluded from analysis (n=0)
Lost to follow-up (n=5)
Discontinued intervention (n=0)
Allocated to Beef intervention
(n= 14)
Received allocated intervention
(n= 9)
Did not receive allocated
intervention (n=0)
Allocated to Whey intervention
(n=14)
Received allocated intervention
(n=9)
Did not receive allocated
intervention (n=0)
Randomized (n=42)
Allocated to CHO group
(n= 14)
Received allocated intervention
(n=9)
Did not receive allocated
intervention (n=0)
Analysed (n= 9)
Excluded from analysis (n =0)
Analysed (n= 9)
Excluded from analysis (n =0)
Lost to follow-up (n=5)
Discontinued intervention (n=0)
Lost to follow-up (n=5)
Discontinued intervention (n=0)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

270 Eur J Appl Physiol (2017) 117:267–277
1 3
Dietary supplementation
The three products under study were presented as 20 g
sachets of vanilla-flavored powder to be diluted in 250 mL
of cold orange juice at each intake. The diluted drinks were
similar in appearance, texture and taste and were isoener-
getic. The nutritional composition of each product and the
amino acids profile of beef and whey proteins are shown in
Table 1. Products were taken once a day for 8 weeks (56
total doses per participant). On training days (resistance
training or recreational exercise sessions), the supplement
was ingested just after training, whereas on non-training
days, the product was administered in the morning, before
having breakfast.
Dietary (nutrition) monitoring
A research nutritionist collected dietary habits and
explained the proper procedures for recording dietary
intake. Each participant completed a 3-day food diary
report (2 weekdays, and 1 weekend day). The food diary
report was then analyzed using Dietplan 6 software to
determine energy and macronutrient content. Participants
were instructed to maintain their normal diet throughout
the training period. To determine changes and evaluate
differences caused by the supplementation protocol, diet
composition was analyzed again during the last week of the
intervention protocol.
Measurements
Body composition
Body mass (BM) and height were assessed, on a standard
scale and stadiometer according the methods described by
Ross and Marfel-Jones (1991).
Whole body densitometry was assessed using air dis-
placement via the Bod Pod® (Life Measurements, Concord,
CA, USA) in accordance with the manufacturer’s instruc-
tions as detailed elsewhere (Dempster and Aitkens 1995).
Briefly, the participants were tested wearing only tight fit-
ting clothing (swimsuit or undergarments) and an acrylic
swim cap. Volunteers wore exactly the same clothing for all
testing. Thoracic gas volume was estimated for all partici-
pants using a predictive equation integral to the Bod Pod®
software. The calculated value for body density was used in
the Siri equation (Siri 1961) to estimate the body composi-
tion. A complete body composition measurement was per-
formed twice. If the agreement on percentage of body fat
was within 0.05%, the two tests were averaged. If the two
tests were not within the 0.05% agreement, a third test was
performed and, then, the average of the three completed tri-
als was used for all body composition variables.
Muscular thickness
Right-side vastus medialis muscle thicknesses were meas-
ured in real time using an Diasus diagnostic ultrasound
imaging unit (Dynamic Imaging, Livingston, Scotland
UK) coupled to a 50 mm probe at a frequency of 7.5 MHZ
while participants were lying supine with arms and legs
completely relaxed. The right lower limb was positioned
with the knee extended. The probe was placed perpendicu-
lar to the skin surface and bone tissues at 80% of the dis-
tance between the lateral condyle of the femur and greater
trochanter (Bradley and O’Donnell 2002). The probe was
coated with a water-soluble transmission gel (Aquasonic
100 Ultrasound Transmission gel) to provide acoustic con-
tact without depressing the dermal surface. Thickness was
calculated as the distance between superficial and deep
aponeuroses measured at the ends and middle region of
each 3.8 cm-wide sonograph.
Three images of each muscle were obtained for each
point and the average of the results was calculated. To
favor reproducibility, probe placement was carefully noted
for reproduction during the other test sessions and the
Table 1 Nutritional composition of drinks per intake (20 g of powder
plus 250 mL of orange juice)
EAA essential amino acids, CHO carbohydrates
Nutrient Beef Whey CHO
Energy value (kcal) 184 179 184
Carbohydrates (g) 25 25 45
Lipids (g) 1.54 0.3 0
Proteins (g) 16.4 18 0
Alanine (g) 1.04 1.06 –
Arginine (g) 1.06 0.38 –
Aspartic acid (g) 1.50 2.29 –
Cysteine (g) 0.16 0.48 –
Glutamic acid (g) 2.58 3.34 –
Glycine (g) 1.07 0.34 –
Histidine (g) 0.55 0.31 –
Isoleucine (g) 0.75 1.00 –
Leucine (g) 1.32 1.93 –
Lysine (g) 1.44 1.81 –
Methionine (g) 0.39 0.44 –
Phenylalanine (g) 0.65 0.61 –
Proline (g) 0.81 1.17 –
Serine (g) 0.65 1.05 –
Threonine (g) 0.73 1.44 –
Tryptophan (g) 0.19 0.39 –
Tyrosine (g) 0.52 5.57 –
Valine (g) 0.80 0.98 –
Total EAA (g) 6.82 8.91 –
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

271Eur J Appl Physiol (2017) 117:267–277
1 3
same operator performed all the measurements. To avoid
any swelling in the muscles that could disturb the results,
images were obtained at least 48 h before and after the pro-
gram intervention.
Training, performance evaluation and control of the
intervention compliance
All participants followed the same resistance training rou-
tine, three times per week alternated with their habitual rec-
reational recreationally training (games or team sport ori-
ented activity) for a total of 8 weeks (24 resistance training
workouts). During the 8 weeks of intervention, participants
carried out their workout session late in the afternoon or
early evening. After a warm up, the participants performed
a total of three circuits involving one set of the following
8 resistance exercises: (1) jump-squat (2) bench press, (3)
parallel back squat, (4) upright row, (5) dumbbell alternate
lunges, (6) shoulder press, (7) lateral hurdle jumps, and (8)
abdominal crunch.
Every exercise included 12 maximum repetitions using
the heaviest possible load (except the abdominal crunch
that involved 20 repetitions per sets). A minimum rest was
permitted in between exercises (only the time required to
change from one exercise to the following). Recovery
between circuits was 2–3 min. Resistance training sessions
were completed in about 30 min. All training sessions were
closely monitored to ensure effort, repetitions and inten-
sity established by experienced strength and conditioning
coaches. All participants completed all lifts for each exer-
cise. The total load (kg) summarized all repetitions from
the first six exercises was considered as the indicator of
performance. Although lateral hurdle jumps and abdomi-
nals were performed with no external load, performing
these exercises at the end of every circuit would impact
on general fitness adaptation and the accumulated level of
fatigue over the training session. The first and last workouts
of the intervention period were used as evaluation sessions.
The participants were instructed to refrain from any vigor-
ous activity for 48 h and avoid caffeine ingestion for at least
48 h prior to both assessment sessions. As occurred during
the entire training period, the first and last sessions were
performed at the same time of the day for the same par-
ticipant. After completing the first session, each participant
was given a batch of products, according to randomization.
Tolerance, collected from adverse events and compli-
ance with product intake (determined by an individual
follow up of the participants), was evaluated continuously
during the intervention. Only the participants who com-
pleted all the 24 training sessions were included in the final
analysis.
Blood indices of health
Blood chemistry samples were taken before and after the
8-week intervention period. Participants arrived at the
physiology laboratory, after a fasting period of 6–8 h, on
two separate occasions (1 day before and 1–2 days after
completing the 8-week intervention period). Finger-prick
capillary whole blood specimens were collected from each
subject, under resting conditions, and analyzed using the
Cholestech LDX Analyzer for fasting total cholesterol,
HDL, LDL cholesterol triglycerides, and glucose, or the
Reflotron Plus Clinical Chemistry Analyzer for CK, cre-
atinine, AST/GOT, ALT/GPT, urea and uric acid. Both
devices were used according to the manufacturer’s instruc-
tions, using test strips (Reflotron Plus Clinical Chemistry
Analyzer) or cassette (Cholestech LDX Analyzer).
Salivary alpha‑defensins HNP1‑3
Saliva collection
Saliva was collected at pre (before and after the first work-
out) and post (before and after the last workout) 8-week
intervention period. At baseline or pre-workout, saliva sam-
ples were collected following the blood samples just a few
minutes before starting the workout. For the post-workout
sample, saliva was collected within 5 min after the exercise
cessation.
Participants remained seated, performing minimal move-
ment for 10 min (pre-workout) or 5 min (post-workout)
prior to each saliva collection. For all saliva samples, the
mouth was rinsed with water at least 5 min before the col-
lection. The participant was requested to swallow to empty
the mouth before each sample collection. Unstimulated
whole saliva was collected by the spitting method while the
participant remained seated, leaning forward and with the
heads tilted down (Navazesh 1993). Saliva was collected
for 1 min. To avoid circadian variation, saliva samples were
collected in between 2 and 7 pm. The collected saliva was
weighed to obtain precise flow rate (g/min) (Dawidson
et al. 1996). The saliva was stored at −80 °C until further
sample treatment and analysis.
Saliva analysis
Saliva samples were centrifuged (12,000g, 10 min; 4 °C)
and the supernatant diluted 1000× with sample dilution
buffer. Each sample was analyzed in duplicate with ELISA
(Hycult biotech, The Netherlands) following the manu-
factures instructions. The calibration curve consisted of
eight standards, ranging from 0.15 to 10 μg/mL HNP1-3.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

272 Eur J Appl Physiol (2017) 117:267–277
1 3
Absorbance (450 nm) values for the saliva samples were
interpolated from calibration standards with a 4-param-
eter logistic curve (My assays, version 2015). In addition,
the alpha-defensins HNP1-3 secretion rates were deter-
mined by multiplying their concentration by the flow rate
(mL min−1).
Statistical analysis
Sensitivity of the final sample size was calculated assum-
ing a model with three groups and four repeated meas-
ures, 0.05 α error probability, and 0.80 power (1 − β), to
ensure adequacy of the study. A descriptive analysis was
performed and subsequently the Kolmogorov–Smirnov
and Shapiro–Wilk test were applied to assess normality.
Sample characteristics at baseline were compared between
conditions (beef vs. whey vs. CHO) using one-way anal-
ysis of variance (ANOVA). Change from pre to post in
performance, body composition, muscle thickness, and
blood indices was calculated by subtracting pre- from post-
values. Differences in change between conditions were
assessed using one-way ANOVA. The changes in the con-
centration and secretion of HNP1-3 or the saliva flow rates
were additionally analyzed using three conditions (beef vs.
whey vs. CHO) × four times (pre-workout 1st vs. post-
workout 1st vs. pre-workout 24th vs. post-workout 24th)
repeated measures ANCOVA, using pre-workout 1st as
covariate. Bonferroni-adjusted post hoc analyses were per-
formed when appropriate. Generalized eta squared (
η2
G
) and
Cohen’s d values were reported to provide an estimate of
standardized effect size (small d = 0.2,
η2
G
= 0.01; mod-
erate d = 0.5,
η
2
G
= 0.06; and large d = 0.8,
η
2
G
= 0.14).
Significance level was set to p < 0.05. Results are reported
as mean ± SD unless stated otherwise. Data analyses were
performed with Stata 13.1 (StataCorp, College Station, TX,
USA).
Results
Fifteen participants (9 male and 6 female) dropped from
the study due to personal reasons, not related with the
intervention protocol. Consequently, 27 participants, 15
males and 12 females (5 (55.5%) and 4 (44.4%) per group,
respectively) successfully completed the study. The sam-
ple was determined to be large enough to detect moderate
group–time interactions (f = 0.26) through a power analy-
sis. The final composition of the three groups was equiva-
lent at baseline (Table 2).
Participants verbally confirmed that they maintained
their diet throughout the trial period. Table 3 shows dietary
monitoring results, presented as the average daily con-
sumption of carbohydrate, protein, fat (g kg−1 day−1), and
energy (kcal kg−1 day−1), before and after intervention
Table 2 Treatment groups
description at baseline
All variables are presented as mean (standard deviation); F and p values were calculated using one-way
analysis of variance with 2/24 degrees of freedom
Variable Beef (n = 9) Whey (n = 9) CHO (n = 9) F(2,24) p
Age (years) 25.6 ± 5.3 27.6 ± 5.2 24.4 ± 7.1 0.63 0.539
Height (m) 1.72 ± 0.09 1.73 ± 0.1 1.71 ± 0.08 0.14 0.872
Body mass (kg) 69.9 ± 14.35 70.27 ± 12.52 70.83 ± 10.43 0.01 0.988
Fat (%) 23.47 ± 11.53 18.87 ± 7.95 23.42 ± 11.65 0.57 0.574
Fat-free mass (%) 76.53 ± 11.53 81.13 ± 7.95 76.58 ± 11.65 0.57 0.574
Fat (kg) 18.54 ± 12.66 13.43 ± 6.9 16.89 ± 10.12 0.59 0.560
Fat-free mass (kg) 52.47 ± 7.43 56.85 ± 10.58 53.94 ± 10.23 0.49 0.618
Vastus Medialis thickness (mm) 28.24 ± 4.59 31.93 ± 3.09 30.04 ± 4.67 1.75 0.194
Total loaded (kg) 7403.4 ± 1746.5 7942.5 ± 1675.7 7215.6 ± 1637.8 0.45 0.643
Table 3 Analysis of the participant’s diet composition
* p < 0.05 significant difference from post- to pre-intervention
Treatment Beef Whey CHO
Pre Post Pre Post Pre Post
Proteins (g kg−1 day−1) 1.45 ± 0.66 1.70 ± 0.70* 1.47 ± 0.75 1.77 ± 0.92* 1.12 ± 0.56 1.12 ± 0.56
CHO (g kg−1 day−1) 3.39 ± 1.5 3.76 ± 1.59* 3.16 ± 2.23 3.55* (2.22) 2.59 ± 0.98 3.20 ± 1.04*
Fat (g kg−1 day−1) 1.20 ± 0.55 1.31 ± 0.56* 1.18 ± 0.40 1.18 ± 0.41 0.91 ± 0.12 0.91 ± 0.12
Energy (kcal kg−1 day−1) 30.99 ± 12.26 33.77 ± 12.41* 29.93 ± 13.50 31.8 ± 13.85* 23.71 ± 7.21 25.83 ± 7.11*
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

273Eur J Appl Physiol (2017) 117:267–277
1 3
for the three treatment groups. At baseline, no between-
group differences were observed for the amount of pro-
tein, carbohydrate, fat or energy intake. However, as a
result of the nutritional intervention, all the three groups
increased the intake of carbohydrate, while the beef and
whey groups raised protein consumption but only beef
increased fat intake. All the three treatment groups signifi-
cantly increased the total energy intake, with no difference
between them.
Body composition, muscle thickness, performance
and blood indices of health
Combining resistance training with any of the nutrition
intervention (beef, whey or CHO) did not produce statis-
tically significant differences between the three treatment
conditions in any of the analyzed variables (Table 4).
However, beef showed large effect sizes for body mass
(d = 1.27); total fat-free mass (d = 0.75) vastus medialis
thickness (d = 1.93) and total kg lifted (d = 2.16); mean-
while, the CHO group showed large effect sizes for the
total kg lifted (d = 1.73) and the vastus medialis thickness
(d = 1.04) and so did the whey treatment only for the total
kg lifted (whey d = 0.97).
Salivary alpha‑defensins HNP1‑3
After adjusting for pre-first-workout using ANCOVA, a
significant supplement–time interaction effect for the con-
centration rates (F[6,72] = 2.47, p = 0.031,
η2
G
= 0.10),
along with statistically significant effect of time
(F[3,72] = 2.69, p = 0.053,
η2
G
= 0.05), but not differences
between treatment conditions (F[2,23] = 0.65, p = 0.533,
η2
G
= 0.014), was observed. No significant interaction
(F(6,72) = 1.43, p = 0.215,
η2
G
= 0.07), treatment condi-
tion (F(2,23) = 0.54, p = 0.591,
η2
G
= 0.008) or time effect
(F(3,72) = 2.01, p = 0.120,
η2
G
= 0.05) was determined on
the secretion rates.
Bonferroni-adjusted post hoc contrasts to baseline
revealed no statistically significant differences between
groups at each time point for both the concentration and
secretion rates. However, the beef treatment condition
showed a statistically significant decrease of the base-
line concentration and secretion rates of HNP1-3 from
the values measured before performing the first workout
(1.90 ± 0.83 μg/mL; 2.95 ± 2.83 μg/min, respectively)
of the values determined at post-intervention, before
(0.92 ± 0.63 μg/mL, p = 0.025, d = 1.03; 0.76 ± 0.74 μg/
min, p = 0.049, d = 0.95), and after (0.95 ± 0.60 μg/
mL, p = 0.032, d = 1.00; 0.59 ± 0.52 μg/min, p = 0.027,
d = 1.02) performing the last workout (Fig. 2a, b).
Saliva flow rate
No significant interaction (F[6,72] = 1.49, p = 0.193,
η2
G
= 0.06) or treatment effect (F[2,24] = 0.47, p = 0.633,
η2
G
= 0.01) was observed. However, a significant time
effect (F[3,72] = 2.92, p = 0.040,
η2
G
= 0.06) was deter-
mined. Post hoc analysis revealed a significant intragroup
decrease for the beef treatment condition in saliva flow rate
Table 4 Differences observed
in body composition, muscle
thickness, performance and
blood markers for the three
treatment conditions after the
8-week intervention period
All variables are presented as mean (standard deviation); F and p values were calculated using one-way
analysis of variance with 2/24 degrees of freedom
Variable Beef (n = 9) Whey (n = 9) CHO (n = 9) F(2,24) p
Body weight (kg) 1.28 ± 1 0.26 ± 1.29 0.45 ± 1.59 1.53 0.236
Fat (%) 0.17 ± 1.35 −0.41 ± 2.08 −0.1 ± 0.44 0.36 0.705
Fat-free mass (%) −0.17 ± 1.35 0.41 ± 2.08 0.1 ± 0.44 0.36 0.705
Fat (kg) 0.47 ± 1.07 −0.33 ± 1.62 0.03 ± 0.77 0.99 0.385
Fat-free mass (kg) 0.88 ± 1.18 0.59 ± 0.86 0.42 ± 0.93 0.49 0.618
Vastus medialis thickness (mm) 3.13 ± 1.62 1.57 ± 3.28 1.7 ± 1.63 1.26 0.300
Total loaded (kg) 853.7 ± 395.2 479.1 ± 494.2 763.2 ± 442.3 1.73 0.198
Total cholesterol (mmol/L) 0.09 ± 0.29 0.06 ± 0.46 −0.26 ± 0.41 2.19 0.134
HDL cholesterol (mmol/L) 0 ± 0.35 0 ± 0.18 −0.11 ± 0.23 0.53 0.593
LDL cholesterol (mmol/L) 0 ± 0.52 0.03 ± 0.5 −0.22 ± 0.17 0.95 0.400
Triglycerides (mmol/L) 0.46 ± 0.98 0.17 ± 0.34 0.14 ± 0.72 0.51 0.608
Glucose (mmol/L) 0.48 ± 1.27 −0.14 ± 0.85 0.27 ± 0.93 0.84 0.445
Urea (mg/dL) 3.7 ± 9.7 2.71 ± 3.64 2.44 ± 7.58 0.07 0.931
Uric acid (mg/dL) −0.33 ± 0.87 −0.18 ± 2.01 0.18 ± 1.06 0.31 0.733
Creatinine (mg) 0.03 ± 0.23 −0.15 ± 0.17 0 ± 0.09 2.61 0.094
AST/GOT (U/L) −0.13 ± 4.1 1.52 ± 5.53 3.43 ± 7.57 0.82 0.451
ALT/GPT (U/L) −0.51 ± 2.57 −1.99 ± 8.12 −2.07 ± 9.25 0.13 0.877
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

274 Eur J Appl Physiol (2017) 117:267–277
1 3
baseline levels from the first to the last training session
at both pre- (1.336 ± 0.752 vs. 0.589 ± 0.398 mL/min,
p = 0.036, d = 0.99) and post-workout (0.928 ± 0.286 vs.
0.470 ± 0.280 mL/min, p = 0.009, d = 0.1.14) sessions.
No significant differences were found between groups at
any time point.
Discussion
Results suggest that consuming a post-workout carbohy-
drate–protein (beef or whey) beverage during an 8-week
resistance training intervention promotes similar training
and body composition outcomes than ingesting only carbo-
hydrate. The most relevant finding of the present investiga-
tion was the decrease of the baseline levels and secretion
rates of the salivary alpha-defensins HNP1-3 determined
after the 8-week intervention protocol in the beef treatment
condition. A similar non-significant trend was observed for
the whey protein group while no changes were reported for
the participants ingesting only carbohydrates. None of the
treatment conditions (beef, whey or CHO) affected the nor-
mal state of the biochemical blood indices of health or elic-
ited acute changes (pre- to post-workout before and after
intervention) of the HNP1-3.
The increase of diet protein consumption in both beef
and whey treatment group did not produce any nega-
tive effects on the analyzed blood markers of metabolism
or hepatic function. The observed response is expected
because the amount of daily protein intake for the both pro-
tein groups was always within the recommended range of
~1–2 g kg−1 (Thomas et al. 2016), Table 3. Indeed, it has
been recently indicated that consuming a high protein diet
(2.6–3.3 g kg−1 day−1) over a 4-month period has no nega-
tive health-related effect on blood lipids or markers of renal
and hepatic function in healthy resistance trained individu-
als (Antonio et al. 2016).
Data from the present investigation indicate that ingest-
ing a post-workout beverage containing protein with carbo-
hydrate or carbohydrate alone did not influence resistance
training responses during the 8-week training period. Over-
all, the three treatment groups showed similar improve-
ments in body composition, muscle mass and the total kg
lifted over the training intervention. However, the beef
group seems to elicit a slightly superior body mass gain
based on fat-free mass accretion. This appreciation would
be supported by the largest effect size observed for the vas-
tus medialis thickness in beef (d = 1.93) compared to the
other two treatment conditions (whey d = 0.48 and CHO
d = 1.04). Even though previous studies have reported
positive effects of canned, minced or beefsteak beef at
promoting training outcomes in young (Negro et al. 2014)
and elderly individuals (Symons et al. 2009), to the best
of the authors’ knowledge, this is the first study to look at
the effect of a hydrolyzed beef protein powder extract and
compare its effects with those elicited using whey protein
and a non-protein isoenergetic nutrient at supporting resist-
ance training outcomes along with analyses of its impact
on health and immune markers in young athletes. The
total energy provided in the three treatment conditions was
almost similar or the amount of protein provided by beef
and whey was not sufficient to elicit remarkable differences
with respect to the contrast non-protein group. Neverthe-
less, the present results would still support the premise that
daily caloric intake appears to be one of the most relevant
factors affecting training adaptations during middle- to
long-term exercise interventions (McLellan et al. 2014).
In contrast to previous studies (Davison et al. 2009;
Gillum et al. 2015), we did not find acute increases of
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
Pre Post Pre Post
HNP1-3 secretion rate (µg/min)
42tuokroW1tuokroW
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Pre PostPre Post
Alpha-defensins HNP1-3 (µg/mL)
42tuokroW1tuokroW
Beef
Whey
CHO
A
B
Fig. 2 Acute and long-term changes in the concentration (a) and
secretion rates (b) of the alpha-defensins HNP1-3 for the three treat-
ment conditions (mean ± 95% confidence intervals). Statistically sig-
nificant differences were only found from the baseline levels meas-
ured at pre- (before the first workout) to post-intervention, measured
at both before and after the last workout for the beef treatment condi-
tion for both the concentration (p = 0.025 and p = 0.049) and secre-
tion rates (p = 0.032 and p = 0.027), respectively
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

275Eur J Appl Physiol (2017) 117:267–277
1 3
HNP1-3 after exercise at both before and after the trained
intervention. Reasons for this discrepancy could be the
nature of the physical exercise. Participants of the present
investigation performed eight different resistance train-
ing exercises while previous studies have used submaxi-
mal cycling (Davison et al. 2009) or running (Gillum et al.
2015) constant intensity protocol. Both aforementioned
studies reported acute increases in the concentration of
HNP1-3 determined immediately after exercise (Davison
et al. 2009; Gillum et al. 2015) or until 1 h compared to
pre-exercise values (Gillum et al. 2015). The post-exercise
rise in the concentration and secretion rate of AMP could
arise from the exercise-induced neutrophilia that occurs in
mucosal secretions potentially due to airway inflammation
or damage to the airway epithelial cells (Muns 1994). In
addition, neutrophilia of the blood can increase HNP1-3 in
saliva (Shiomi et al. 1993). These effects have been asso-
ciated with endurance cyclic exercises such as running or
cycling but not with resistance type exercises. Neutrophils
contain HNP1-3 in addition to other AMP, such as Lacto-
ferrin and Lysozyme (Gleeson 2007). Endurance continu-
ous exercise has been shown to activate neutrophils pos-
sibly causing the release of their contents into the saliva
(Pyne 1994), and this is likely the cause of increased sali-
vary levels of AMP after exercise. Even though participants
of the current investigation were instructed to perform 12
repetitions of each exercise using the maximum possible
amount of load and, consequently, all of them experienced
maximum level of fatigue at the end of every session, no
acute increase in the salivary concentration of HNP1-3 was
determined for both at the beginning and at the end of the
8-week intervention period. Perhaps, the different nature of
the applied training protocol alternating exercises involving
different muscle groups could have influenced the observed
response.
It has been reported that an initial reduction in the con-
centration of some salivary AMP may be an adaptive
response to exercise (West et al. 2010). However, the initial
decrease would not be observed in previously well training
athletes. Participants of the present investigation were rec-
reationally but regular resistance training individuals and
only the participants included in the protein groups (beef
and whey, respectively) showed a significant or a trend
to decrease the levels of HNP1-3. The aforementioned
response would reflect a reduced immune competence with
a concomitant increase in the susceptibility to opportunis-
tic infection that has been reported in well-trained athletes
(West et al. 2010). However, the clinical relevance of the
above-mentioned modification is unknown, since the lev-
els of HNP1-3 measured for the three treatment conditions
were similar to those obtained in previous studies (Davison
et al. 2009; Kunz et al. 2015). In fact, the salivary levels of
AMP including HNP1-3 can vary about 100-fold between
individuals. It is, therefore, difficult to define normal-base-
line reference ranges (Gorr 2009). Nonetheless, when the
individual responses are compared, it is worth noting that
all participants but one in the beef group depicted a con-
sistent decrease in the levels of the HNP1-3 from pre- to
post-intervention, whereas those in the whey and CHO
condition showed different patterns of responses with
three participants in the whey and two in the CHO treat-
ment group showing a reduced concentration of HNP1-3
post-intervention.
The HNP1-3 entail a specific group of alpha-defensins
that would increase in response to dental caries and the
respiratory distress syndrome (Kunz et al. 2015). The after
intervention decreased level of HNP1-3 observed in the beef
and whey treatment groups could be related to the reduced
concentration of carbohydrate ingested during the post-
workout period (0.36 ± 0.08 and 0.37 ± 0.06 g kg−1 for the
beef and whey groups, respectively, vs. 0.65 ± 0.10 g kg−1
for the CHO treatment condition). These results would sup-
port the notion that only carbohydrate ingestion with no
added proteins would probably be the only effective nutri-
tional countermeasure to exercise-induced acute immune
dysfunction (Walsh et al. 2011a). Nonetheless, the effec-
tiveness of ingesting carbohydrate at blunting the post-
exercises immunosuppression has been mainly observed
in acute studies (Walsh et al. 2011a) where the decrease
of different immunological markers including some sali-
vary defensins could be interpreted as a transient immune
dysfunction with a concomitant higher risk of infection or
virus attack (Davison et al. 2009). However, as the drop in
the HNP1-3 level observed in the present study was not
produced acutely but after an 8-week intervention, it can-
not be analyzed as a transient response but as an adaptive
medium- to long-term adaptation similar to that observed
in other salivary markers in well-trained athletes (West
et al. 2010). Considering that HNP1-3 are a marker associ-
ated with exercise stress (Kunz et al. 2015), the lower level
observed after the intervention in the beef group would be
interpreted as a positive adaptive response that allows indi-
viduals to reduce the level of stress determined by a given
exercise protocol. As previously mentioned, neutrophils are
the most abundant white blood cell type and HNP1-3 are
the most abundant antimicrobial peptide secreted by neu-
trophils. Thus, decreased levels of HNP1-3 have also been
proposed as an index of reduced risk factor against virus
and infection (Mehlotra et al. 2016).
The decreased levels of HNP1-3 resulted after the
8-week intervention program, when participants achieved a
better level of performance, which is in line with previous
studies that have reported a lower concentration of salivary
AMP, including HNP1-3 in better-fit athletes (Kunz et al.
2015). Therefore, the reduced HNP1-3 levels observed
after 8 weeks of training could be considered as a normal
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

276 Eur J Appl Physiol (2017) 117:267–277
1 3
adaptive response that might have been hasten by the inges-
tion of the beef protein beverage. Individual differences in
training adaptation can be the reason by which some par-
ticipants allocated in the whey and CHO condition showed
a similar post-intervention reduction in the HNP1-3 levels.
Additionally, the lower average concentration of HNP1-3
was also related to an increased salivary flow rate observed
only for the beef treatment condition. However, differently
from the study of Kunz et al., no differences in perfor-
mance were observed between the three conditions (beef,
whey, and CHO) compared to the present study. The higher
salivary flow rate, observed in the beef group, may act as
a compensatory mechanism to maintain adequate rates of
salivary AMP secretion and may, in fact, be indicative of
greater parasympathetic nervous system activity (resulting
in a large amount of diluted saliva) and vagal tone under
the similar training stimulus (Chicharro et al. 1998; Coote
2010) for this particular group.
It is worthy to note that the large variability in the con-
centration of HNP1-3 observed at rest and in response to
exercise observed in the current study is in accordance
with others (Davison et al. 2009) who have recognized
the variance as a source of limitation for the detection of
intervention-induced changes in mucosal parameters, par-
ticularly in a parallel group design (Jones et al. 2015).
Given the importance of mucosal immune parameters
toward host defense (West et al. 2010), further investigation
of the effects of post-workout feeding strategies on other
salivary AMP (e.g., salivary Lactoferrin and Lysozyme) is
warranted.
In conclusion, the present investigation suggests that the
ingestion of a carbohydrate–protein (beef or whey) post-
workout beverage may support some possible adaptations
induced by resistance training in a comparable way as the
ingestion of only carbohydrate. Furthermore, the ingestion
of 20 g of protein powder, mainly from hydrolyzed beef
protein, would also promote an early decrease of the aver-
age baseline levels and secretion rates of the salivary alpha-
defensins (HNP1-3) with no negative effect on the blood
indices of health in recreationally trained college athletes.
Acknowledgements Funding was provided by (MEATPROT).
Compliance with ethical standards
Funding MEATPROT and the University of Greenwich provided
joint funding for the completion of this project; however, this does not
affect this original research content and purpose.
Conflict of interest The authors declare that they have no conflicts of
interest relevant to the content of this manuscript.
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution 4.0 International License (http://crea-
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction 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.
References
Antonio J, Ellerbroek A, Silver T, Vargas L, Peacock C (2016) The
effects of a high protein diet on indices of health and body com-
position—a crossover trial in resistance-trained men. J Int Soc
Sports Nutr 13:3. doi:10.1186/s12970-016-0114-2
Bradley M, O’Donnell P (2002) Atlas of musculoskeletal ultrasound
anatomy Greenwich Medical Media London
Chernoff R (2004) Protein and older adults. J Am Coll Nutr
23:627S–630S
Chicharro JL, Lucia A, Perez M, Vaquero AF, Urena R (1998) Saliva
composition and exercise. Sports Med 26:17–27
Coote JH (2010) Recovery of heart rate following intense
dynamic exercise. Exp Physiol 95:431–440. doi:10.1113/
expphysiol.2009.047548
Cruzat VF, Krause M, Newsholme P (2014) Amino acid supplementa-
tion and impact on immune function in the context of exercise. J
Int Soc Sports Nutr 11:61. doi:10.1186/s12970-014-0061-8
Davison G, Allgrove J, Gleeson M (2009) Salivary antimicrobial
peptides (LL-37 and alpha-defensins HNP1-3), antimicrobial
and IgA responses to prolonged exercise. Eur J Appl Physiol
106:277–284. doi:10.1007/s00421-009-1020-y
Dawidson IJ, Ar’Rajab A, Melone LD, Poole T, Griffin D, Risser
R (1996) Early use of the Gore-Tex Stretch Graft. Blood Purif
14:337–344
Dempster P, Aitkens S (1995) A new air displacement method for
the determination of human body composition. Med Sci Sports
Exerc 27:1692–1697
Gillum TL, Kuennen MR, Castillo MN, Williams NL, Jordan-Pat-
terson AT (2015) Exercise, but not acute sleep loss, increases
salivary antimicrobial protein secretion. J Strength Cond Res
29:1359–1366. doi:10.1519/JSC.0000000000000828
Gleeson M (2000) Mucosal immune responses and risk of respiratory
illness in elite athletes. Exerc Immunol Rev 6:5–42
Gleeson M (2007) Immune function in sport and exercise. J Appl
Physiol 103(2):693–699. doi:10.1152/japplphysiol.00008.2007
Gorr SU (2009) Antimicrobial peptides of the oral cavity. Periodontol
2000(51):152–180. doi:10.1111/j.1600-0757.2009.00310.x
Jones AW, Thatcher R, March DS, Davison G (2015) Influence of
4 weeks of bovine colostrum supplementation on neutrophil and
mucosal immune responses to prolonged cycling. Scand J Med
Sci Sports 25:788–796. doi:10.1111/sms.12433
Kunz H et al (2015) Fitness level impacts salivary antimicrobial pro-
tein responses to a single bout of cycling exercise. Eur J Appl
Physiol 115:1015–1027. doi:10.1007/s00421-014-3082-8
McLellan TM, Pasiakos SM, Lieberman HR (2014) Effects of pro-
tein in combination with carbohydrate supplements on acute or
repeat endurance exercise performance: a systematic review.
Sports Med 44:535–550. doi:10.1007/s40279-013-0133-y
Mehlotra RK, Zimmerman PA, Weinberg A (2016) Defensin gene
variation and HIV/AIDS: a comprehensive perspective needed. J
Leukoc Biol 99:687–692. doi:10.1189/jlb.6RU1215-560R
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

277Eur J Appl Physiol (2017) 117:267–277
1 3
Morton RW, McGlory C, Phillips SM (2015) Nutritional interventions
to augment resistance training-induced skeletal muscle hypertro-
phy Front Physiol 6:245. doi:10.3389/fphys.2015.00245
Muns G (1994) Effect of long-distance running on polymorphonu-
clear neutrophil phagocytic function of the upper airways. Int J
Sports Med 15:96–99. doi:10.1055/s-2007-1021027
Naclerio F, Larumbe-Zabala E, Cooper R, Allgrove J, Earnest CP
(2015) A multi-ingredient containing carbohydrate, proteins
l
-glutamine and
l
-carnitine attenuates fatigue perception with
no effect on performance, muscle damage or immunity in soc-
cer players. PLoS One 10:e0125188. doi:10.1371/journal.
pone.0125188
Navazesh M (1993) Methods for collecting saliva. Ann N Y Acad Sci
694:72–77
Negro M, Vandoni M, Ottobrini S, Codrons E, Correale L, Buono-
core D, Marzatico F (2014) Protein supplementation with low fat
meat after resistance training: effects on body composition and
strength. Nutrients 6:3040–3049. doi:10.3390/nu6083040
Nieman DC, Bishop NC (2006) Nutritional strategies to counter stress
to the immune system in athletes, with special reference to foot-
ball. J Sports Sci 24:763–772
Pyne DB (1994) Regulation of neutrophil function during exercise.
Sports Med 17:245–258
Reidy PT, Rasmussen BB (2016) Role of ingested amino acids and
protein in the promotion of resistance exercise-induced muscle
protein anabolism. J Nutr. doi:10.3945/jn.114.203208
Rennie MJ, Wackerhage H, Spangenburg EE, Booth FW (2004) Con-
trol of the size of the human muscle mass. Annu Rev Physiol
66:799–828. doi:10.1146/annurev.physiol.66.052102.134444
Ross WD, Marflel-Jones MJ (1991) Kineanthropometry. In: MacDou-
gal JC, Wenger HA, Green HJ (eds) Physiological testing of high
performance athlete. Human Kinetics, Champaign, pp 223–308
Shiomi K, Nakazato M, Ihi T, Kangawa K, Matsuo H, Matsukura
S (1993) Establishment of radioimmunoassay for human
neutrophil peptides and their increases in plasma and neutrophil
in infection. Biochem Biophys Res Commun 195:1336–1344.
doi:10.1006/bbrc.1993.2190
Siri WE (1961) Body composition from fluid spaces and density:
analysis of methods. In: Brozek J, Henschel A (eds) Techniques
for measuring body composition. National Academy of Sciences,
National Research Council, Washington, D.C., pp 223–244
Symons TB, Sheffield-Moore M, Wolfe RR, Paddon-Jones D (2009)
A moderate serving of high-quality protein maximally stimu-
lates skeletal muscle protein synthesis in young and elderly
subjects. J Am Diet Assoc 109:1582–1586. doi:10.1016/j.
jada.2009.06.369
Thomas DT, Erdman KA, Burke LM (2016) Position of the Acad-
emy of Nutrition and Dietetics, Dietitians of Canada, and the
American College of Sports Medicine: Nutrition and Athletic
Performance. J Acad Nutr Diet 116:501–528. doi:10.1016/j.
jand.2015.12.006
Walsh NP et al (2011a) Position statement. Part two: Maintaining
immune health. Exerc Immunol Rev 17:64–103
Walsh NP et al (2011b) Position statement. Part one: Immune func-
tion and exercise. Exerc Immunol Rev 17:6–63
Wang G (2014) Human antimicrobial peptides and proteins. Pharma-
ceuticals (Basel) 7:545–594. doi:10.3390/ph7050545
West NP, Pyne DB, Kyd JM, Renshaw GM, Fricker PA, Cripps AW
(2010) The effect of exercise on innate mucosal immunity. Br J
Sports Med 44:227–231. doi:10.1136/bjsm.2008.046532
Wilkinson SB, Tarnopolsky MA, Macdonald MJ, Macdonald JR,
Armstrong D, Phillips SM (2007) Consumption of fluid skim
milk promotes greater muscle protein accretion after resistance
exercise than does consumption of an isonitrogenous and isoen-
ergetic soy-protein beverage. Am J Clin Nutr 85:1031–1040
Yount NY, Yeaman MR (2012) Emerging themes and therapeutic
prospects for anti-infective peptides. Annu Rev Pharmacol Toxi-
col 52:337–360. doi:10.1146/annurev-pharmtox-010611-134535
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com