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Creatine Supplementation Does Not Influence the Ratio
Between Intracellular Water and Skeletal Muscle Mass
in Resistance-Trained Men
Alex S. Ribeiro
University of Northern Paraná
Ademar Avelar
State University of Maringá
Witalo Kassiano and João
Pedro Nunes
Londrina State University
Brad J. Schoenfeld
CUNY Lehman College
Andreo F. Aguiar
University of Northern Paraná
Michele C.C. Trindade
State University of Maringá
Analiza M. Silva and Luís B. Sardinha
Universidade de Lisboa
Edilson S. Cyrino
Londrina State University
The authors aimed to compare the effects of creatine (Cr) supplementation combined with resistance training on skeletal muscle
mass (SMM), total body water, intracellular water (ICW), and extracellular water (ECW) in resistance-trained men as well as to
determine whether the SMM/ICW ratio changes in response to the use of this ergogenic aid. Twenty-seven resistance-trained men
received either Cr (n= 14) or placebo (n= 13) over 8 weeks. During the same period, subjects performed two split resistance
training routines four times per week. SMM was estimated from appendicular lean soft tissue assessed by dual-energy X-ray
absorptiometry. Total body water, ICW, and ECW were determined by spectral bioelectrical impedance. Both groups showed
improvements (p<.05) in SMM, total body water, and ICW, with greater values observed for the Cr group compared with
placebo. ECW increased similarly in both groups (p<.05). The SMM/ICW ratio did not change in either group (p>.05), whereas
the SMM/ECW ratio decreased only in the Cr group (p<.05). A positive correlation was observed (p<.05) between SMM and
ICW changes (r= .71). The authors’results suggest that the increase in muscle mass induced by Cr combined with resistance
training occurs without alteration of the ratio of ICW to SMM in resistance-trained men.
Keywords:DXA, ergogenic aids, muscle hypertrophy, strength training
Muscle hypertrophy is one of the main morphological adapta-
tions resulting from resistance training (RT). Due to the trainability
principle, trained individuals exhibit a lesser magnitude of gains in
muscle mass than novice or detrained subjects (Ahtiainen et al.,
2003;Kraemer & Ratamess, 2004). That is why trained individuals
often resort to the use of ergogenic strategies to overcome plateaus
and continue to realize meaningful adaptations during an RT
program (Helms et al., 2014). Among the plethora of ergogenic
options, creatine (Cr) supplementation when combined with RT
generally has been shown as an effective aid to enhance muscle
mass accretion (Chilibeck et al., 2017;Nissen & Sharp, 2003),
particularly in trained individuals (Nunes et al., 2017).
Among the mechanisms by which Cr supplementation can
contribute to muscle mass gain, early research proposed that this
substance appears to directly affect muscle protein synthesis via
modulations of components in the mammalian target of the rapa-
mycin pathway (Farshidfar et al., 2017). Cr supplementation may
directly affect the myogenic process by altering secretions of
myokines (e.g., myostatin and insulin-like growth factor-1) and
the expression of myogenic regulatory factors, resulting in
enhanced satellite cells’mitotic activities and differentiation into
myonuclei (Farshidfar et al., 2017). Moreover, Cr acts as an
osmolyte, inducing an increase in intracellular water (ICW), which,
in turn, may increase protein synthesis and, thus, contribute to
muscle mass accretion (Farshidfar et al., 2017;Schoenfeld, 2010).
The simultaneous analysis of changes in total body water (TBW)
and its extracellular water (ECW) and ICW components may help
explain the changes that occur in skeletal muscle mass (SMM) in
response to Cr supplementation combined with RT.
Results of previous investigations have been inconsistent as
to the effects of Cr supplementation combined with RT in relative
changes in aspects of body composition. Francaux and Poortmans
(1999) observed that Cr supplementation combined with RT
induced an increase in absolute values of body mass, TBW, and
ICW but not in ECW in untrained men. However, when TBW and
ICW were expressed as a percentage of body mass, no significant
difference was observed. It should be noted that the experiment
mentioned above was conducted with untrained individuals.
Ribeiro and Aguiar are with the Center for Research in Health Sciences, University of
Northern Paraná, Londrina, Paraná, Brazil. Avelar and Trindade are with the Depart-
ment of Physical Education, State University of Maringá, Maringá, Paraná, Brazil.
Kassiano, Nunes, and Cyrino are with the Metabolism, Nutrition, and Exercise
Laboratory, Physical Education and SportCenter, LondrinaState University, Londrina,
Paraná, Brazil. Schoenfeld is with the Exercise Science Department, CUNY Lehman
College, Bronx, New York, NY, USA. Silva and Sardinha are with the Exercise and
Health Laboratory, CIPER, Faculdade Motricidade Humana, Universidade de Lisboa,
Lisbon, Portugal. Kassiano (witalo.o liveira@uel.br) is corresponding author.
1
International Journal of Sport Nutrition and Exercise Metabolism, (Ahead of Print)
https://doi.org/10.1123/ijsnem.2020-0080
© 2020 Human Kinetics, Inc. ORIGINAL RESEARCH
Therefore, considering that the adaptive response to RT is, at least
in part, predicated on training status (Ahtiainen et al., 2003), the
ability to compare muscular adaptations in untrained individuals
may be confounded by the fact that they tend to respond favorably
to multiple training stimuli. Moreover, the Francaux and Poort-
mans (1999) study analyzed relative changes using body mass,
which is not an accurate indicator of muscular adaptations.
Powers et al. (2003) revealed that Cr supplementation com-
bined with RT apparently did not induce a significant increase in
ICW, but there was an increase in TBW. It is, therefore, conceiv-
able that the increased TBW was due to ECW, although the values
were not reported in the study. These results may have been
influenced by the inclusion of both men and women in the same
sample as well as the response to a different routine in trained
individuals. For example, recently, it has been observed that a
program that individualizes prescription based on previous training
volume elicits significantly greater changes in the cross-sectional
area than a standard RT program in resistance-trained men
(Scarpelli et al., 2020). Therefore, the internal validity of studies
involving individuals with previous RT experience would seem-
ingly benefit from standardizing RT variables among a cohort for a
prolonged period before initiating the research protocol.
In this regard, our investigation aimed to compare the effects of
RT with Cr supplementation versus placebo (Pla) on SMM, TBW,
ICW, and ECW in resistance-trained men who followed a similar
preconditioning-RT protocol and the same progression in training
volume before the study onset. We also analyzed whether differences
occurred in the ICW/SMM ratio from Cr supplementation after 8
weeks of RT. Based on previous studies, we hypothesized that the Cr
group would present a higher SMM gain than the Pla group. However,
the ratio of ICW to SMM would remain similar within groups.
Methods
Experimental Design
A randomized, double-blinded, and Pla-controlled trial was carried
out over 30 weeks. Participants and outcome assessors are all
unaware of the intervention assignments during the trial. Testing
was carried out at Weeks 1–2, 19–20, and 29–30 and consisted of
anthropometric and body composition measurements. A precondi-
tioning RT period was performed during Weeks 3–18 to equalize the
training conditions between subjects. Following the preconditioning
period, the participants were submitted at the 8-week intervention
with RT plus Cr or Pla supplementation (Weeks 21–28). During this
period, dietary intake was monitored over the first and last weeks of
intervention (Weeks 21 and 28). Participants were allocated through
random number generation. The first measurements took place
1 week before the start of training. The second and third evaluations
started on the following Monday and Tuesday after the end of
each training phase (Friday), allowing 48–72 hr between the last
training and body composition assessment. Figure 1presents the
experimental design.
Participants
Thirty men were recruited through social media and personal
invitations to participate in this study. Of these, three dropped out
before finishing the intervention due to personal reasons or insuffi-
cient training frequency. Thus, a total of 27 men from a university-
based population participated in our study and performed both the
preconditioning and the supplementation phase. Participants were
included in the study if they had no reported disease symptoms, no
orthopedic injuries, were not vegetarian/vegan, were not using any
nutritional supplements (i.e., protein and Cr powders), were not
using anti-inflammatory medicine, and declared that they were free
from the use of anabolic steroids. Written informed consent was
obtained from all participants after receiving a detailed description of
the study procedures. This investigation was conducted according to
the Declaration of Helsinki and was approved by the Londrina State
University Ethics Committee (protocol number: 028/2012).
Anthropometry
Body mass was measured to the nearest 0.1 kg using a calibrated
electronic scale (Balmak, Laboratory Equipment Labstore, Cur-
itiba, Paraná, Brazil) with the participants wearing light workout
clothing and no shoes. Height was measured with a stadiometer
attached to the scale to the nearest 0.1 cm with subjects standing
shoeless. Body mass index was calculated as body mass (in kilograms)
divided by the square of height (in meters).
Body Composition
Body composition assessment was conducted in the morning hours.
Participants were instructed to urinate ∼30 min before the evaluation,
Figure 1 —Experimental design. Cr = creatine; Pla = placebo; RT = resistance training.
(Ahead of Print)
2Ribeiro et al.
refrain from ingesting food or drink in the previous 4 hr, avoid
strenuous physical exercise for at least 24 hr, refrain from consump-
tion of alcoholic and caffeinated beverages for at least 48 hr, and
avoid the use of diuretics for at least 7 days prior each assessment.
A spectral bioelectrical impedance device (Xitron Hydra, model
4200; Xitron Technologies Inc., San Diego, CA) was used to
estimate TBW, ICW, and ECW. Participants removed all metal
objects such as earrings, watches, chains, bracelets from the body
before the measurements began. Measurements were performed on a
table that was isolated from electrical conductors with subjects lying
supine along the table’s longitudinal centerline axis, legs abducted at
an angle of 45° relative to the body midline, and hands pronated.
After cleaning the skin with alcohol, two electrodes were placed on
the surface of the right hand and two on the right foot by procedures
described by Sardinha et al. (1998). The spectral bioelectrical
impedance device was calibrated each day according to the man-
ufacturer’s recommendations. The same professional performed the
exams in the pre- and postintervention periods, and the intraclass
correlation coefficient (ICC) and standard error of measurement
(SEM) were: SEM of 0.19 l and ICC = .99 for ICW, SEM of 0.32 l
and ICC = .98 for ECW, and SEM of 0.38 l and ICC = .98 for TBW.
Whole-body dual-energy X-ray absorptiometry (Lunar Prod-
igy, model NRL41990; GE Lunar, Madison, WI) was used to assess
appendicular lean soft tissue and body fat. The SMM was estimated
by the predictive equation proposed by Kim et al. (2004) as follows:
SMM (in kilograms) = (appendicular lean soft tissue ×1.19) –1.65.
Participants removed all metal objects such as earrings, watches,
chains, bracelets from the body before the exams began. Scans were
performed with the subjects lying in the supine position along the
table’s longitudinal centerline axis. Feet were taped together at the
toes to immobilize the legs while the hands were maintained in a
pronated position within the scanning region. Subjects remained
motionless during the entire scanning procedure. Both calibration
and analysis were carried out by a skilled laboratory technician.
Equipment calibration followed the manufacturer’s recommenda-
tions. The software generated standard lines that set apart the limbs
from the trunk and head. These lines were adjusted by the same
technician using specific anatomical points determined by the
manufacturer. Analyses during the intervention were performed
by the same technician, who was blinded to group identity through-
out the investigation. The ICC and SEM for SMM were .98 and
0.29 kg, respectively.
Supplementation Protocol
During the supplementation period, the participants were randomly
divided into two groups (Pla or Cr supplementation). The supple-
mentation with Cr included a loading period (5 days of 20 g),
consumed in four daily doses, followed by a maintenance period
(51 days of 3 g), consumed in one dose at breakfast. Maltodextrin
was used as a Pla with the same doses and procedures employed in
the Cr group. The capsules of Cr or Pla were given to the partici-
pants with the exact number of units for the 8 weeks of supple-
mentation. The intake of the supplementation was encouraged by
the researchers throughout the study period. Every training day, the
participants were asked about their adherence to supplementation
and whether they felt any side effects related to its consumption.
Dietary Intake
Participants were instructed by a nutritionist to complete a food
record on three nonconsecutive days (two weekdays and one
weekend day). Subjects were given specific instructions regarding
the recording of portion sizes and quantities to identify all food and
fluid intake. Total dietary energy, protein, carbohydrate, and fat
content were calculated using Avanutri Processor Nutrition soft-
ware, version 3.1.4 (Avanutri Equipamentos de Avaliação Ltda,
Três Rios, RJ, Brazil). All subjects were asked to maintain their
regular diet throughout the study period.
Progressive RT
The progressive RT program was divided into three phases of 8
weeks in a manner designed to induce muscular hypertrophy. All
participants were personally supervised by physical education
professionals throughout each training session to reduce deviations
from the study protocol and to ensure participant safety. During
the first two phases, all participants performed a single progressive
RT program three times per week on nonconsecutive days
(Monday, Wednesday, and Friday), consisting of exercises for the
upper limbs, trunk, and lower limbs. Three sets of 8–12 repetition
maximum were performed for each exercise. Subjects were in-
structed to perform each repetition with a 1-s concentric phase
followed by a 2-s eccentric phase. The rest period between sets
lasted 60–90 s, with a 2–3 min interval between each exercise.
Subjects were encouraged to exert maximal effort on all sets. When
12 repetitions were completed for all sets, the training load was
increased by 2–5% for upper body exercises and 5–10% for lower
body exercises for the next training session.
The first phase of the progressive RT program consisted of
nine exercises performed in the following order: (1) bench press,
(2) leg press, (3) wide-grip behind-the-neck pulldown, (4) leg
extension, (5) side lateral raise, (6) lying leg curl, (7) triceps
pushdown, (8) calf press on the leg press machine, and (9) arm
curl. In the second phase, the training program was altered, and 11
exercises were performed in the following order: (1) bench press,
(2) incline dumbbell fly, (3) wide-grip behind-the-neck pulldown,
(4) seated cable rows, (5) seated barbell military press, (6) arm curl,
(7) lying triceps press, (8) leg extension, (9) leg press, (10) lying
leg curl, and (11) seated calf raise. After the resistance exercises,
the abdominal crunch exercise was performed on the floor using
the subject’s body mass (three sets of 30–50 repetitions in both
phases).
In the third phase, wherein participants received supplementa-
tion, the RT sessions were performed four times per week, divided
into two routines (A and B) wherein Program A was executed on
Mondays and Thursdays and was composed of exercises for the
chest, shoulders, triceps, and abdominal muscles in the following
order: (1) bench press, (2) inclined dumbbell fly, (3) cable cross
over, (4) barbell military press, (5) lateral raise, (6) upright row,
(7) lying triceps French press, (8) triceps pushdown, and (9) crunch.
Program B was conducted on Tuesdays and Fridays, incorporating
exercises for the back, biceps, forearm, thigh, and calves in the
following order: (1) wide-grip lat pulldown, (2) seated cable row,
(3) arm curl, (4) alternating dumbbell curl, (5) wrist curl, (6) squat
on a Smith machine, (8) knee extension, (9) leg curl, and (10) seated
calf raise.
During this third phase, the subjects performed four sets for all
exercises with the load increasing and the number of repetitions
simultaneously decreasing for each set (pyramid system). Thus, the
number of repetitions used in each set was 12/10/8/6 repetition
maximum, respectively, except for the calves (15–20 repetition
maximum) and abdominal muscles (150–300 repetitions per ses-
sion). The load was increased for each set by 2–4 kg for upper
(Ahead of Print)
Creatine and Hypertrophy in Resistance-Trained Men 3
body exercises and 3–6 kg for lower body exercises. The load
progression was planned so that when the participant was able
to perform two more repetitions in the last set for a given exercise
on two consecutive sessions, the load for the next session was
increased 2–5% for the exercises of the upper limbs and 5–10% for
the exercises of the lower limbs. Subjects were instructed to
perform each repetition with a concentric to eccentric phase ratio
of 1:2. The rest period between sets lasted 1–2 min, with a 2–3min
rest interval between each exercise. Adherence to the program was
satisfactory with all subjects completing >85% of the total sessions.
Statistical Analysis
Two-way analyses of variance for repeated measures were per-
formed for all comparisons. When the Fratio was significant,
Fisher’s post hoc test was employed to identify where mean
differences existed. An independent student’sttest was performed
to determine whether baseline values were significantly different
between groups and to compare the percentage changes. The
Spearman correlation coefficient was used to determine the rela-
tionship between percentage changes in SMM and percentage
changes in ICW and ECW. The effect size (ES) was calculated
as posttraining mean minus pretraining mean divided by the pooled
pretraining SD (Cohen, 1992). Relative ES was calculated as the
ES difference between conditions (Cr ES −Pla ES). An ES of 0.20–
0.49 was considered as small, 0.50–0.79 as moderate, and ≥0.80 as
large (Cohen, 1992). For all statistical analyses, significance was
accepted at p<.05. The data were analyzed using SPSS software
(version 20.0; SPSS Inc., Chicago, IL).
Results
Table 1displays the general characteristics of the participants
immediately before the presupplementation phase. No differences
were observed between groups (p>.05). The macronutrients and
energy intake at different time points of the study are presented in
Table 2. There was no significant (p>.05) effect for a group by
time interaction and for the main effect of the group for any of the
nutritional outcomes analyzed, indicating no between-group dif-
ferences in the first and last week of training. However, a significant
main effect of time (p<.05) was observed for protein, carbohy-
drate, and energy intake, meaning that both groups increased the
consumption of these macronutrients throughout the study period.
The body composition values at pre- and postsupplementation
phase are presented in Table 3. An interaction was found for
body mass, SMM, TBW, and ICW (p<.05), wherein increases
were greater in the Cr group compared with Pla in body mass
(Cr = +3.0%, ES = 0.27; Pla = +0.7%, ES = 0.06), SMM (Cr =
+7.1%, ES = 0.71; Pla = +2.8%, ES = 0.28), TBW (Cr = +7.0%,
ES = 0.68; Pla = +1.7%, ES = 0.16), and ICW (Cr = +9.2%,
ES = 0.81; Pla = +1.6%, ES = 0.14). An effect of time was observed
for ECW and percentage of body hydration (p<.05), with both
groups reaching a similar increase for ECW (Cr = +1.2%,
ES = 0.13; Pla = +0.6%, ES = 0.06) and percentage body hydration
(Cr = +3.6%, ES = 0.58; Pla = +1.1%, ES = 0.18). The ratio
between ICW and SMM did not reach statistical significance
(p>.05) for any main effects; however, the ratio between ECW
and SMM decreased only in the Cr group. No significant effect was
observed for body fat (p>.05).
Figure 2displays percentage changes from the pre- to post-
supplementation phase in SMM, ICW, and ECW according to
group. Increases were higher for Cr than Pla for SMM and
ICW (p<.05).
A correlation between percentage changes in SMM and ICW
was observed (r= .71, p<.001), indicating that the higher the
change in SMM, the greater the change in ICW. However, no
significant correlation was observed between percentage changes
in SMM and ECW (r= .12, p>.05). Figure 3displays the correla-
tion between percentage change from pre- to posttraining in SMM
and ICW.
Discussion
The present investigationwas designed to investigate the effects of Cr
supplementation on changes in SMM, TBW, and its ICW and ECW
components when combined with an 8-week RT program. We also
endeavored to determine whether this ergogenic aid could influence
the ICW/SMM and ECW/SMM ratios in resistance-trained men. The
main findings of the present study were: (a) the increase in SMM,
ICW, and TBW was higher in the Cr group when compared with Pla;
(b) the ICW/SMM ratio did not change due to Cr supplementation
combined with RT in resistance-trained men; (c) there was a 50%
shared variance between ICW and SMM; and (d) groups did not
differ in ECW and percentage body water responses.
Consistent with the current literature, our findings highlight
the effectiveness of Cr supplementation in enhancing RT-induced
Table 1 General Characteristics of the Sample
Variables
Creatine
(n= 14)
Placebo
(n= 13) p
Age (years) 21.8 ± 4.1 21.7 ± 4.2 .99
Body mass (kg) 70.1 ± 7.4 68.0 ± 8.4 .48
Height (cm) 173.0 ± 4.5 175.5 ± 7.3 .30
Body mass index (kg/m
2
) 23.3 ± 2.1 22.0 ± 2.3 .13
Note. Data are presented as mean ± SD.
Table 2 Energy and Macronutrients Intake at Different Moments According to Group
Creatine (n= 14) Placebo (n= 13) Effects
Variables Pre Post Pre Post Group Time Interaction
Energy (kcal·kg
−1
·day
−1
) 28.65 ± 7.3 31.25 ± 10.0* 27.36 ± 8.2 35.44 ± 10.1* .65 .03 .27
Protein (g·kg
−1
·day
−1
) 1.28 ± 0.4 1.51 ± 0.6* 1.22 ± 0.4 1.67 ± 0.6* .79 .03 .46
Carbohydrate (g·kg
−1
·day
−1
) 3.94 ± 1.4 4.02 ± 1.5* 3.88 ± 1.2 4.92 ± 1.37* .48 .04 .89
Lipid (g·kg
−1
·day
−1
) 0.93 ± 0.2 1.01 ± 0.3 0.93 ± 0.3 1.00 ± 0.3 .99 .38 .93
Note. Data are presented as mean ± SD.
*p<.05 versus pre.
(Ahead of Print)
4Ribeiro et al.
gains in muscle mass (Kerksick et al., 2018). Among the proposed
mechanisms for the effects of Cr supplementation in promoting
muscle mass accretion, an increase in intracellular hydration has
been furthered as a likely contributor. Cr is cotransported with Na
+
ions across the sarcolemma, which initiates an influx of Cl
−
and
water to balance electroneutrality and osmolality (Odoom et al.,
1996), thus contributing to the movement of ECW to the ICW
compartment. Moreover, Cr has been shown to enhance glycogen
storage (van Loon et al., 2004). This may further heighten intra-
cellular hydration, given that each gram of glycogen attracts
approximately 3–4 g of water (Olsson & Saltin, 1970). The
observed increase in SMM in both groups, particularly in the
group supplemented with Cr, was associated with ICW expansion.
This association is expected as ICW may be regarded as a
functional water compartment. There is substantial evidence to
suggest that cellular hydration status is an essential factor
controlling cellular protein turnover (Häussinger et al., 1993). In
fact, cell swelling acts as an anabolic signaling stimulus, mediating
muscle protein synthesis via the mitogen-activated protein kinase
signaling cascade (Niisato et al., 1999). Moreover, Silva et al.
(2014) found that athletes who increase ICW over the season
improved power and strength, suggesting that ICW may be an
indicator of performance.
The more significant increase in ICW from Cr supplementa-
tion combined with RT has been previously reported by Francaux
and Poortmans (1999) in a cohort of untrained men submitted to
9 weeks of supplementation or Pla. Alternatively, it has also been
reported that Cr supplementation combined with RT did not induce
a significant increase in ICW in a cohort of resistance-trained men
and women (Powers et al., 2003). The reason that our results differ
from this latter investigation is not clear. It may be, at least in part,
related to the fact that we employed an adaptation phase wherein
all participants performed the same program for 16 weeks, whereas
Figure 2 —Percentage of changes from pre- to posttraining (8 weeks)
on SMM, ICW, and ECW in resistance-trained men according to groups (Pla,
n=13; Cr, n= 14). Data are expressed as mean and SD. SMM = skeletal
muscle mass; Cr = creatine; Pla = placebo; ICW = intracellular water; ECW =
extracellular water. *p<.05 versus Pla.
Table 3 Participants’Scores at Pre and Post the 8-Week Intervention Period
Creatine (n= 14) Placebo (n= 13) Relative
effect size
Relative
differences
Interaction
pvalueVariables Pre Post Pre Post
Body mass (kg) 70.1 ± 7.4 72.2 ± 6.9 67.9 ± 8.4 68.4 ± 8.1 0.20 2.3 <.01
Appendicular lean soft tissue (kg) 28.7 ± 2.5 30.6 ± 2.6* 28.3 ± 4.0 29.1 ± 4.0* 0.43 4.3 <.001
SMM (kg) 32.5 ± 2.5 34.8 ± 2.6*,** 32.1 ± 4.0 33.0 ± 4.0* 0.43 4.3 <.001
Body fat (%) 13.8 ± 5.7 14.0 ± 5.8 12.5 ± 6.5 12.8 ± 6.1 −0.02 −1.0 .93
Total body water (L) 43.0 ± 3.1 46.0 ± 3.3*,** 41.8 ± 5.7 42.5 ± 4.9* 0.52 5.3 <.01
ICW (L) 26.2 ± 2.1 28.6 ± 2.4*,** 25.3 ± 3.8 25.7 ± 3.2* 0.68 7.6 <.01
ECW (L) 17.1 ± 1.1 17.3 ± 1.2* 16.7 ± 2.1 16.8 ± 1.9* 0.06 0.6 .87
ICW/SMM (L/kg) 0.81 ± 0.06 0.82 ± 0.05 0.79 ± 0.07 0.78 ± 0.61 0.31 2.5 .14
ECW/SMM (L/kg) 0.54 ± 0.02 0.52 ± 0.02* 0.53 ± 0.01 0.53 ± 0.02 −1.00 −4.8 <.001
Percentage of body water 61.7 ± 3.2 63.9 ± 4.1* 61.5 ± 4.4 62.2 ± 4.1* 0.39 2.4 .10
Note. Data are expressed as mean ± SD. Effect size classification = 0.20–0.49 small, 0.50–0.79 moderate, and ≥0.80 large. Relative effect size = effect size of creatine group
minus effect size of Pla group; relative differences = Δ% of creatine group minus Δ% of Pla group; ICW = intracellular water; ECW = extracellular water; SMM = skeletal
muscle mass.
*p<.05 versus pre. **p<.05 versus Pla.
Figure 3 —Association between percentage changes from pre- to post-
training (8 weeks) on SMM and ICW in resistance-trained men (n= 27).
SMM = skeletal muscle mass; ICW = intracellular water.
(Ahead of Print)
Creatine and Hypertrophy in Resistance-Trained Men 5
the previous study initiated the study period immediately upon
consent. It is conceivable that novel aspects of the training protocol
confounded results in the study by Powers et al. (2003) and, thus,
masked potential changes in ICW between conditions; the novelty
factor would not apply to our study given the imposed acclimation
phase. This hypothesis warrants further investigation.
Despite the greater ICW increase in the Cr group compared
with Pla, the ICW/SMM ratio did not change in any group. We did
not attempt to measure intramuscular Cr stores, but similar loading/
maintenance protocols have shown these storage depots to be
significantly elevated after supplementation (Preen et al., 2003).
Given that Cr acts as an osmolyte, it can be surmised that, perhaps,
similar amounts of contractile hypertrophy occurred between
conditions and that the additional increases in SMM observed in
the Cr group were attributed mainly to “sarcoplasmic hypertrophy”
as previously described in the literature (Haun, Vann, Osburn,
et al., 2019). Further research is warranted by employing the biopsy
technique to determine subfractional changes within the muscle
when Cr supplementation is combined with regimented RT.
Another important finding of our investigation is that Cr
supplementation did not promote changes in ECW. Despite the
lack of effect, the RT protocol did elicit an increase in this water
compartment. This result may be related to the rise of SMM,
because the water in the skeletal muscle is distributed to both ICW
and ECW (the sum of interstitial fluid and blood plasma; Haun,
Vann, Roberts, et al., 2019). Thus, an increased SMM is seemingly
accompanied by increased ECW and ICW. Hence, it is reasonable
to speculate that the increases in SMM were associated with an
expanded extracellular fluid balance.
It is noteworthy that the ECW/SMM ratio decreased only in
the Cr group. Recently, it has been reported that the relative
expansion of ECW above that of ICW may have an adverse effect
on muscle quality (Yamada et al., 2017). Based on this information,
our finding of a reduced ECW/SMM ratio following Cr supple-
mentation compared with Pla may confer a beneficial effect on
SMM quality. This hypothesis warrants further study.
To the best of our knowledge, this is the first investigation to
analyze the response of SMM and ICW to the combination of RT
and Cr supplementation in resistance-trained men. Although our
study has notable strengths—(a) a randomized, double-blinded,
and Pla-controlled trial; (b) a preconditioning-RT protocol to
reduce confounding from novelty; and (c) validated testing mea-
sures—it is worth mentioning some possible limitations. First, we
did not prescribe a dietary protocol for participants throughout the
experiment but, rather, only monitored dietary consumption during
the first and last weeks of training. Therefore, we were unable to
verify whether the diets they followed may have unduly affected
our findings. However, all participants were asked continuously not
to change their eating habits during our experiment, and random
group assignment should, conceivably, diminish potential con-
founding in this regard. Moreover, pre- and poststudy nutritional
assessment did not reveal significant between-group differences
in energy or macronutrient intake. Second, the duration of the
supplementation phase of our investigation was relatively short
(8 weeks). Therefore, it is not clear whether these responses would
occur similarly after a longer period of RT and Cr supplementation.
We should also note that dual-energy X-ray absorptiometry scans
were performed at least 4 hr after fluid and food ingestion. An
overnight fast would be the most effective way to minimize
measurement errors in lean soft tissue determination (Nana et al.,
2015). Also, biopsies would have been beneficial to help assure that
Cr supplementation was successfully administrated. Finally, our
study is specific to resistance-trained young men and should not
necessarily be generalized to other populations, including women,
youth, and older individuals.
Conclusion
This investigation suggests that Cr supplementation combined with
RT induces higher increases in SMM and ICW but does not change
the ratio between SMM and ICW in resistance-trained young men.
These inferences’strength of confidence must be balanced with the
limitations mentioned above of the study design. From a practical
standpoint, our results reinforce that Cr supplementation combined
with RT is an effective ergogenic aid that promotes increased
muscle mass in resistance-trained men. According to our findings,
changes in SMM and ICW occur similarly, with no differences in
the ratio between these two components, even with Cr supplemen-
tation. Therefore, resistance-trained men who use Cr will be able to
maximize muscle mass gains, accompanied by increased cellular
hydration, when combined with RT.
Acknowledgments
The authors thank the individuals who participated in this study. The
Coordination of Improvement of Higher Education Personnel (CAPES/
Brazil) conferred scholarship to W. Kassiano (MSc) and J.P. Nunes (MSc),
and the National Council of Technological and Scientific Development
(CNPq/Brazil) conceded grants to E.S. Cyrino. The study was designed by
E.S. Cyrino, A.S. Ribeiro, B.J. Schoenfeld, and A. Avelar; data were
collected and analyzed by W. Kassiano, J.P. Nunes, M.C.C. Trindade, and
A. Avelar; data interpretation and manuscript preparation were undertaken
by A.S. Ribeiro, B.J. Schoenfeld, W. Kassiano, A.M. Silva, and L.B.
Sardinha. All authors approved the final version of the paper. The author
B.J. Schoenfeld serves on the scientific advisory board for Dymatize
Nutrition, a manufacturer of sport supplements. The other authors declare
no conflict of interest.
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