Journal of Strength and Conditioning Research, 2006, 20(4), 928–931
? 2006 National Strength & Conditioning Association
EFFECTS OF TWENTY-EIGHT DAYS OF BETA-ALANINE
AND CREATINE MONOHYDRATE SUPPLEMENTATION ON
THE PHYSICAL WORKING CAPACITY AT
NEUROMUSCULAR FATIGUE THRESHOLD
JEFFREY R. STOUT,1JOEL T. CRAMER,2MICHELLE MIELKE,3JOSEPH O’KROY,2DON J. TOROK,2
AND ROBERT F. ZOELLER1
1Department of Health and Exercise Science, University of Oklahoma, Norman, Oklahoma 73019;2Department of
Exercise Science and Health Promotion, Florida Atlantic University, Davie, Florida 33314;3Department of
Nutrition and Health Sciences, Center for Youth Fitness and Sports Research, University of Nebraska–Lincoln,
Lincoln, Nebraska 68588.
ABSTRACT. Stout, J.R., J.T. Cramer, M. Mielke, J. O’Kroy, D.J.
Torok, and R.F. Zoeller. Effects of twenty-eight days of beta-
alanine and creatine monohydrate supplementation on the phys-
ical working capacity at neuromuscular fatigue threshold. J.
Strength Cond. Res. 20(4):928–931. 2006.—The purpose of this
study was to examine the effects of 28 days of beta-alanine (b-
Ala) and creatine monohydrate (CrM) supplementation on the
onset of neuromuscular fatigue by using the physical working
capacity at neuromuscular fatigue threshold (PWCFT) test in un-
trained men. Fifty-one men (mean age ? SD ? 24.5 ? 5.3 years)
volunteered to participate in this 28-day, double-blind, placebo-
controlled study and were randomly assigned to 1 of 4 groups:
placebo (PLA; 34 g dextrose; n ? 13), CrM (5.25 g CrM plus 34
g dextrose; n ? 12), b-Ala (1.6 g b-Ala plus 34 g of dextrose; n
? 12), or b-Ala plus CrM (CrBA; 5.25 g CrM plus 1.6 g b-Ala
plus 34 g dextrose; n ? 14). The supplement was ingested 4
times per day for 6 consecutive days, then twice per day for 22
days before posttesting. Before and after the supplementation,
subjects performed a continuous incremental cycle ergometry
test while a surface electromyographic signal was recorded from
the vastus lateralis muscle to determine PWCFT. The adjusted
mean posttest PWCFTvalues (covaried for pretest PWCFTvalues)
for the b-Ala and CrBA groups were greater than those for the
PLA group (p ? 0.05). However, there were no differences be-
tween the CrM vs. PLA, CrBA vs. b-Ala, CrM vs. b-Ala, or CrM
vs. CrBA groups (p ? 0.05). These findings suggested that b-Ala
supplementation may delay the onset of neuromuscular fatigue.
Furthermore, there appeared to be no additive or unique effects
of CrM vs. b-Ala alone on PWCFT.
KEY WORDS. carnosine, ergogenic aids, electromyography, cycle
plitude and fatigue during submaximal cycle ergometry
to identify the power output that corresponds to the onset
of neuromuscular fatigue. The PWCFT represents the
highest power output that results in a nonsignificant (p
? 0.05) increase in muscle activation of the vastus later-
alis over time. The PWCFTtest has been shown to be re-
liable (2, 4, 17), valid (2), and sensitive to changes in fit-
ness level (2); however, the physiological mechanism re-
sponsible for the increase in EMG amplitude during a
eVries et al. (3, 4) developed an incremental cycle
ergometer test called the physical working capacity
at fatigue threshold (PWCFT), which utilizes the re-
lationship between electromyographic (EMG) am-
fatiguing task is unknown. Two potential mechanisms in-
clude the accumulation of metabolic by-products (lactate,
hydrogen ions [H?], inorganic phosphate (Pi), and am-
monia) and the depletion of stored energy substrates
(adenosine triphosphate, phosphocreatine [PCr], and gly-
cogen) (13). It has been suggested that skeletal muscle
PCr may serve as a temporal energy buffer as well as a
modulator of glycolysis, and that, if increased via creatine
supplementation (12), it may influence neuromuscular fa-
tigue (16, 21). In support of this hypothesis, Stout et al.
(16) demonstrated that 5 days of creatine monohydrate
(CrM) loading (4 ? 5 g·d?1) in trained women’s crew team
members significantly (p ? 0.05) increased PWCFT. To
date, however, no studies have examined the effects of
CrM supplementation on PWCFTin men.
Recent studies by Hill et al. (10) and Harris et al. (8)
have demonstrated that 28 days of beta-alanine (b-Ala;
4–6 g·d?1) supplementation increased intramuscular lev-
els of carnosine by approximately 60%. It has been sug-
gested that carnosine serves as a buffer and helps main-
tain skeletal muscle acid-base homeostasis when a large
quantity of H?is produced during high-intensity exercise
(19). Harris et al. (7) demonstrated improvements in per-
formance during a 4-minute maximal cycle ergometry
test in men after supplementing with b-Ala (3.2 g·d?1) for
5 weeks. The authors concluded that the improvements
may have been caused by an enhanced H?buffering ca-
pacity as a result of increased muscle carnosine levels
after b-Ala supplementation (7).
In theory, increasing skeletal muscle PCr and carno-
sine concentrations via CrM and b-Ala supplementation,
respectively, will work synergistically to delay fatigue by
decreasing the reliance on anaerobic glycolysis, reducing
intramuscular lactate accumulation, and buffering H?
during incremental cycle ergometry. However, no previ-
ous studies have examined both the unique and the com-
bined effects of CrM and b-Ala supplementation on neu-
romuscular fatigue. The purpose of this study, therefore,
was to examine the effects of 28 days of b-Ala and CrM
supplementation on the onset of neuromuscular fatigue
as measured by the PWCFTtest in untrained men.
Experimental Approach to the Problem
None of the subjects had ingested creatine, or any other
dietary supplements, for a minimum of 12 weeks before
EFFECTS OF BETA-ALANINE AND CREATINE ON FATIGUE
physical working capacity at fatigue threshold (PWCFT) for 1
subject. EMG ?Vrms ? electromyographic amplitude in root
mean squared microvolts; NS ? not significant.
Illustration of the method used to determine the
the initiation of the study. During the course of the study,
the subjects were asked to maintain their current exer-
cise and dietary patterns and to abstain from other nu-
tritional supplements, nonprescription drugs, and caf-
feine. After pretesting, the subjects were randomly as-
signed to 1 of 4 treatment conditions using a double-blind
design: (a) placebo (PLA; 34 g dextrose; n ? 13), (b) cre-
atine (CrM; 5.25 g CrM plus 34 g dextrose; n ? 12), (c)
b-Ala (1.6 g b-Ala plus 34 g dextrose; n ? 12), or (d) b-
Ala plus CrM (CrBA; 5.25 g of CrM plus 1.6 g b-Ala plus
34 g dextrose; n ? 14). The supplements were identical
in taste and appearance, and were dissolved in 16 oz of
water and ingested 4 times per day for 6 consecutive days,
then twice per day for 22 days before posttesting. Third-
party random laboratory testing was conducted on the
supplement packets, and the contents were determined
to be accurate.
Fifty-one men (mean age ? SD ? 24.5 ? 5.3 years, height
? 171.9 ? 27.9 cm, and weight ? 82.0 ? 7.1 kg) volun-
teered for this investigation. All procedures were ap-
proved by the Institutional Review Board before the ini-
tiation of the study, and each subject was advised of any
possible risks before providing informed consent.
A bipolar (2.54-cm center-to-center) surface electrode
(Quinton Quick Prep silver–silver chloride; Quinton In-
strument Co., Bothell, WA) arrangement was placed on
the right thigh over the lateral portion of the vastus la-
teralis muscle, midway between the greater trochanter
and the lateral condyle of the femur. The reference elec-
trode was placed over the iliac crest. Interelectrode im-
pedance was kept below 2,000 ? by careful abrasion of
the skin. The raw EMG signals were preamplified (gain:
?1,000) using a differential amplifier (EMG100C; Biopac
Systems, Inc., Santa Barbara, CA), sampled at 1,000 Hz,
and stored on a personal computer for off-line analysis.
The EMG signals were later band-pass filtered from 10
to 500 Hz (2nd-order Butterworth filter) and expressed as
root mean square amplitude values by software (Acq-
Knowledge version 3.7; Biopac).
Determination of Physical Working Capacity at
The PWCFTvalues were determined from the vastus la-
teralis muscle using the methods described by deVries et
al. (3, 4) (Figure 1). The subjects began pedaling (with toe
clips) at 60 W (70 rpm) on a calibrated, electronically-
braked cycle ergometer (Lode Excalibur Sport Cycle Er-
gometer, Groningen, The Netherlands). The power output
was then increased by 30 W every 2 minutes until the
subject could no longer maintain 70 rpm. During each 2-
minute interval, six 10-second EMG samples were re-
corded from the vastus lateralis. The PWCFTwas deter-
mined by averaging the highest power output that re-
sulted in a nonsignificant (p ? 0.05; single-tailed t-test)
slope value for the EMG amplitude vs. time relationship
with the lowest power output that resulted in a signifi-
cant (p ? 0.05) slope value (Figure 1).
Test-retest reliability for the PWCFTtest was deter-
mined by using a separate sample of 12 subjects mea-
sured 28 days apart. Using the recommendations of Weir
(22), the intraclass correlation coefficient (r) was 0.948
(SEM ? 22.79 W), which was similar to values reported
by Stout et al. (16) and deVries et al. (2, 3) in young ath-
letic women (r ? 0.94), young men (r ? 0.947), and older
men (r ? 0.976). In addition, there was no significant dif-
ference (p ? 0.721) between the mean PWCFTvalues from
test 1 (mean ? SEM ? 204.2 ? 18.1 W) to test 2 (201.9
? 20.9 W).
Two separate 1-way analyses of covariance (ANCOVA)
were used to analyze the PWCFTdata based on the rec-
ommendations of Huck and McLean (11). The indepen-
dent variable, group, included 4 levels: PLA, b-Ala, CrM,
and CrBA. The pretest and posttest values were used as
the covariate and dependent variable, respectively. Pre-
liminary least squares regression analyses were conduct-
ed to examine the linearity of the relationships between
the covariate and the dependent variable within all
groups, and the interaction between the covariate and
group was used to test for homogeneity of slopes (6).
When appropriate, Bonferroni-corrected post hoc pair-
wise comparisons were used to examine the differences
among the groups. For effect size, the partial eta squared
(?2) statistic was calculated, and according to Green et al.
(6), ?2of 0.01, 0.06, and 0.14 represents small, medium,
and large effect sizes, respectively. An alpha of p ? 0.05
was established a priori. SPSS (version 11.5, SPSS, Inc.,
Chicago, IL) was used for all statistical analyses.
Table 1 contains the mean and SEM values for the pre-
testing and posttesting PWCFTresults. There were sig-
930STOUT, CRAMER, MIELKE ET AL.
TABLE 1. Mean and SEM values for PWCFT(W) at pretesting
and posttesting for each group.*
adjusted for the initial differences in pretest PWCFT(covari-
ate). *The increase in PWCFTfrom pretesting to posttesting
was greater for the beta-alanine group than for the placebo
group (p? 0.004, ?2? 0.17). †The increase in PWCFTfrom pre-
testing to posttesting was greater for the creatine ? beta-ala-
nine group than for the placebo group (p? 0.011, ?2? 0.13).
Means values (? SEM) for posttest PWCFTscores
nificant linear relationships between pretest PWCFTand
PLA (p ? 0.001, r ? 0.95, slope ? 1.18), b-Ala (p ? 0.001,
r ? 0.91, slope ? 1.14), CrM (p ? 0.001, r ? 0.92, slope
? 1.17), and CrBA (p ? 0.001, r ? 0.82, slope ? 0.83)
groups. There was no interaction (p ? 0.123, ?2? 0.12)
between pretest PWCFTand group, which supported the
homogeneity-of-slopes assumption. The posttest PWCFT
means were adjusted during the ANCOVA procedure
based on the pretest PWCFT differences for the PLA
(?29.0 W), b-Ala (?18.6 W), CrM (?16.0 W), and CrBA
(?2.9 W) groups. The ANCOVA indicated a significant
difference (p ? 0.019, ?2? 0.19) among the group means
for the posttest PWCFTvalues after adjusting for the pre-
test differences. The strength of association (i.e., effect
size, ?2) indicated that the treatment groups (PLA, b-Ala,
CrM, and CrBA) accounted for 19% of the variance of the
posttest PWCFT values, holding constant the pretest
PWCFTscores. Bonferroni-corrected pairwise comparisons
indicated that the increase in PWCFTfrom pretesting to
posttesting was greater for the b-Ala group than for the
PLA group (p ? 0.004, ?2? 0.17), and the increase from
pretesting to posttesting was greater for the CrBA group
than for the placebo group (p ? 0.011, ?2? 0.13). There
were no differences between the CrM and PLA groups (p
? 0.136, ?2? 0.05), b-Ala and CrM groups (p ? 0.132, ?2
? 0.05), b-Ala and CrBA groups (p ? 0.591, ?2? 0.01),
or CrM and CrBA groups (p ? 0.305, ?2? 0.02). Figure
2 shows the group means (? SEM) for the posttest PWCFT
scores adjusted for the initial differences in pretest
The primary and original findings of this study suggested
that b-Ala supplementation may delay the onset of neu-
romuscular fatigue during incremental cycle ergometry.
Furthermore, there appeared to be no unique or additive
effects of CrM on PWCFTcompared to b-Ala alone. In
agreement, Hill et al. (10) examined the effects of CrM
and/or b-Ala supplementation on work completed during
cycling to exhaustion at 110% of estimated power maxi-
mum in men. The authors reported that 28 days of sup-
plementing b-Ala or CrM increased the amount of work
completed; however, there appeared to be no additive ef-
fect when both were supplemented simultaneously (10).
McClaren et al. (13) have suggested that a decrease
in muscle pH, as a result of the accumulation of H?or
intracellular and extracellular ammonia, may be respon-
sible for fatigue-induced increases in muscle activation
and the corresponding increase in EMG amplitude. Tay-
lor et al. (20) also found that, for incremental cycle er-
gometry, the accumulation of plasma lactate and ammo-
nia was associated with an increase in EMG amplitude
measured from the rectus femoris muscle. Therefore, ev-
idence has suggested that a reliance on anaerobic glycol-
ysis and the subsequent H?accumulation that ensues
leads to an increase in EMG amplitude from the working
muscles because of elevated lactate concentractions and
decreases in pH.
In the present study, 28 days of b-Ala supplementa-
tion resulted in a significant increase in PWCFT(b-Ala ?
14.5%; CrBA ? 11%); this may have been caused by an
increase in carnosine concentrations, which may have en-
hanced intramuscular H?buffering capacity (7, 8, 10, 19).
Harris et al. (7, 8) and Hill et al. (10) have hypothesized
that increasing muscle carnosine through b-Ala supple-
mentation will help maintain the intramuscular environ-
ment during intensive exercise by countering the accu-
mulation of H?. The results of the present study support-
ed this hypothesis and suggested that b-Ala supplemen-
tation may delay the fatigue-induced increase in EMG
amplitude during submaximal cycle ergometry, which
may occur as a result of carnosine-induced increases in
H?buffering capacity. Future studies should measure
muscle carnosine and lactate concentrations during sub-
maximal fatiguing exercise with and without prior b-Ala
supplementation to verify this hypothesis.
Few studies have been conducted to determine the ef-
fect of CrM supplementation on submaximal exercise per-
formance. Nelson et al. (14) reported that CrM loading in
athletic men and women (age range 21–27 years) resulted
in a 12% increase in ventilatory threshold as well as a
decrease in blood lactate and ammonia concentrations
during incremental cycle ergometry. In addition, Stout et
al. (17) reported that CrM loading in women’s crew ath-
letes (age range 18–21 years) resulted in a significant (p
? 0.05) increase (13%) in PWCFT. Several investigators
(5, 14, 15, 16, 21) have hypothesized that increasing mus-
cle PCr content by CrM supplementation may decrease
the reliance on anaerobic glycolysis, reduce intramuscu-
lar lactate accumulation, and consequently delay the on-
set of fatigue.
In the present study, 28 days of CrM supplementation
resulted in an 11.3% increase in PWCFT, which was sim-
ilar to the 13% increase observed in our previous study
(17). However, the increase in PWCFTfor the CrM group
EFFECTS OF BETA-ALANINE AND CREATINE ON FATIGUE
reported in the present study was not statistically differ-
ent (p ? 0.05) from the change reported in the PLA group.
In support of these findings, Stroud et al. (18) reported
that CrM supplementation had no effect on respiratory
gas exchange or blood lactate accumulation during an in-
cremental treadmill test in physically active men. It is
possible that the discrepancies regarding the effects of
CrM supplementation on performance are related to the
variability in muscle creatine retention as a result of CrM
loading (1, 9). Casey et al. (1) demonstrated a positive
relationship (r ? 0.71, p ? 0.05) between anaerobic ex-
ercise performance during cycle ergometry and the mag-
nitude of muscle creatine retention as a result of CrM
supplementation. It was concluded that the improve-
ments in anaerobic performance were dependent on the
magnitude of muscle creatine retention following CrM
In summary, the b-Ala and CrBA groups had signifi-
cantly greater PWCFTvalues when compared to the PLA
group, which indicated that 28 days of b-Ala supplemen-
tation (with or without CrM) may delay the onset of neu-
romuscular fatigue during incremental cycle ergometry in
men. This delay in neuromuscular fatigue may have been
caused by the augmented muscle carnosine levels, which
may have resulted in a greater capacity to buffer H?dur-
ing exercise. However, there appeared to be no unique or
additive effects of CrM on PWCFTvs. b-Ala alone. To test
this hypothesis, future studies should examine muscle
carnosine and lactate concentrations during submaximal
fatiguing exercise with and without b-Ala and/or CrM
The primary results of this study suggested that b-Ala
supplementation (3.2 g·d?1) for 28 days may delay the on-
set of neuromuscular fatigue and improve physical work-
ing capacity during cycle ergometry. Although recommen-
dations must await further clinical trials, these findings
may be useful for nutritionists, strength and conditioning
professionals, and athletes contemplating the use of sup-
plemental b-Ala. In addition, these findings may provide
a foundation for future studies to test the hypothesis that
b-Ala supplementation may increase muscle carnosine
concentrations, which consequently may enhance the H?
buffering capacity within skeletal muscle.
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This study was funded by a grant from Experimental and Ap-
plied Sciences, Inc., Golden, CO. We would also like to thank Dr.
Sue B. Graves and Marni P. Rakes for there assistance in man-
aging this study.