Short-Duration Beta-Alanine Supplementation Did Not Prevent the Detrimental Effects of an Intense Preparatory Period on Exercise Capacity in Top-Level Female Footballers

Abstract

Purpose: High-intensity activity is an important aspect of football performance during competitive match play. The aim of this study was to investigate the effect of beta-alanine supplementation throughout a short-duration intense football-specific training period prior to an international competition on measures of high-intensity running performance. Methods: Twenty-four elite international U20 female footballers (age 18 ± 1 y, height 1.67 ± 0.07 m, body mass 62.7 ± 7.4 kg) volunteered to perform the YoYo Intermittent Recovery Test Level 1 (YoYo IR1), the Running Anaerobic Sprint Test (RAST) and a 20-m maximal sprint test on two separate occasions, separated by 3 weeks of training and supplementation. Participants were randomly assigned to receive either 6.4 g·day−1 sustained-release beta-alanine (BA, N = 12) or an equivalent dose of maltodextrin (placebo, PL, N = 12) throughout a 3-week standardized training camp. Results: There was a main effect of group (P = 0.05) and time (P = 0.004) on YoYo IR1; overall values were lower in PL and distance covered was lower post- vs. pre-supplementation. There was no group × time interaction (P = 0.07). There was an effect of sprint number for RAST, but no further main effects and there were no effect for the 20-m sprint. Conclusions: Top-level female footballers involved in this intense 3-week training period prior to a competition worsened their high-intensity intermittent exercise capacity, and this negative result was not attenuated by a short-duration BA supplementation protocol throughout the same period. Further work is necessary to elucidate whether adapted training protocols and BA dosing regimens could lead to better results.

Full text

ORIGINAL RESEARCH
published: 21 April 2020
doi: 10.3389/fnut.2020.00043
Frontiers in Nutrition | www.frontiersin.org 1April 2020 | Volume 7 | Article 43
Edited by:
David Christopher Nieman,
Appalachian State University,
United States
Reviewed by:
David Hooper,
Jacksonville University, United States
Sophie C. Killer,
English Institute of Sport,
United Kingdom
*Correspondence:
Bryan Saunders
drbryansaunders@outlook.com
Specialty section:
This article was submitted to
Sport and Exercise Nutrition,
a section of the journal
Frontiers in Nutrition
Received: 13 January 2020
Accepted: 20 March 2020
Published: 21 April 2020
Citation:
Ribeiro R, Duarte B, Guedes da
Silva A, Ramos GP, Rossi Picanço A,
Penna EM, Coswig V, Barbalho M,
Gentil P, Gualano B and Saunders B
(2020) Short-Duration Beta-Alanine
Supplementation Did Not Prevent the
Detrimental Effects of an Intense
Preparatory Period on Exercise
Capacity in Top-Level Female
Footballers. Front. Nutr. 7:43.
doi: 10.3389/fnut.2020.00043
Short-Duration Beta-Alanine
Supplementation Did Not Prevent the
Detrimental Effects of an Intense
Preparatory Period on Exercise
Capacity in Top-Level Female
Footballers
Rafael Ribeiro 1, Breno Duarte 1, André Guedes da Silva 1, Guilherme Passos Ramos 2,
Andreia Rossi Picanço 2, Eduardo Macedo Penna 3, Victor Coswig 3, Matheus Barbalho 4,
Paulo Gentil 4, Bruno Gualano 1and Bryan Saunders 1,5
*
1Applied Physiology and Nutrition Research Group, School of Physical Education and Sport, Rheumatology Division,
Faculdade de Medicina FMUSP, Universidade de São Paulo, São Paulo, Brazil, 2Confederação Brasileira de Futebol, Rio de
Janeiro, Brazil, 3Physical Education, Federal University of Pará – Campus Castanhal, Castanhal, Brazil, 4Physical Education
and Dance Institute, Federal University of Goiás, Goiânia, Brazil, 5Institute of Orthopaedics and Traumatology, Faculty of
Medicine FMUSP, University of São Paulo, São Paulo, Brazil
Purpose: High-intensity activity is an important aspect of football performance during
competitive match play. The aim of this study was to investigate the effect of beta-alanine
supplementation throughout a short-duration intense football-specific training period
prior to an international competition on measures of high-intensity running performance.
Methods: Twenty-four elite international U20 female footballers (age 18 ±1 y, height
1.67 ±0.07 m, body mass 62.7 ±7.4 kg) volunteered to perform the YoYo Intermittent
Recovery Test Level 1 (YoYo IR1), the Running Anaerobic Sprint Test (RAST) and a 20-m
maximal sprint test on two separate occasions, separated by 3 weeks of training and
supplementation. Participants were randomly assigned to receive either 6.4 g·day−1
sustained-release beta-alanine (BA, N=12) or an equivalent dose of maltodextrin
(placebo, PL, N=12) throughout a 3-week standardized training camp.
Results: There was a main effect of group (P=0.05) and time (P=0.004) on YoYo
IR1; overall values were lower in PL and distance covered was lower post- vs. pre-
supplementation. There was no group ×time interaction (P=0.07). There was an effect
of sprint number for RAST, but no further main effects and there were no effect for the
20-m sprint.
Conclusions: Top-level female footballers involved in this intense 3-week training period
prior to a competition worsened their high-intensity intermittent exercise capacity, and
this negative result was not attenuated by a short-duration BA supplementation protocol
throughout the same period. Further work is necessary to elucidate whether adapted
training protocols and BA dosing regimens could lead to better results.
Keywords: football training, nutritional supplementation, YoYo intermittent recovery test, repeated sprints,
competition, fatigue, elite

Ribeiro et al. Short-Duration Beta-Alanine Supplementation Female Footballers
INTRODUCTION
Football, also commonly termed as soccer, is the world’s most
popular sport, practiced by men and women all around the
world (1). The women’s game has seen a stark increase in
both popularity and professionalism over the past decade,
with research into the physiological demands of the women’s
game following a similar rise in popularity (2). The general
characteristics of women’s football demonstrate that match-
play is predominantly performed at low-intensity activities
interspersed by numerous high-intensity actions throughout
(2,3). Key moments that can affect the outcome of a game
generally occur at high-intensities. High-intensity efforts are
reduced during various phases of international matches and vary
according to position (4) and top-level women players have
been shown to perform more high-intensity running and sprints
during games than their less successful counterparts (5). High-
intensity activity, therefore, appears to be an important aspect of
football performance during competitive match play.
Several field tests are employed to evaluate the training
status of football players and are commonly used to predict
match performance and determine the effectiveness of a training
intervention. The YoYo Intermittent Recovery Tests (Level 1
[YoYo IR1] and 2 [YoYo IR2]) evaluate an individual’s capacity
to repeatedly perform and recover from intense exercise bouts,
and is applicable to team sports players due to the specificity of
the exercise undertaken (6). The Yo-Yo IR1 and IR2 have been
shown to correlate to various variables of match performance
and can be used as an indicator of the physical performance
of elite female players throughout competitive matches (7,8),
making them appropriate models to examine the effect of any
intervention designed to manipulate changes in performance
during team sports. In addition to differentiating between playing
standard, these tests can be used to monitor training adaptations,
seasonal variation and determine differences between playing
position [for review, see (9)]. The running anaerobic sprint test
(RAST) is another protocol that has been shown to be reliable
and valid to assess anaerobic power and is a good predictor of
short-distance running performances (10) while the 20-m sprint
test is a commonly used measure to assess team sport players (11).
Any changes in these performance measures may be reflective of
an enhanced capacity to improve in-match performance and thus
are useful tools to determine the efficacy of any intervention.
The preparation period prior to an international competition
is a delicate one in which fitness training must ensure a
maintenance and rebuilding process following an intense season
to ensure peak condition for the subsequent intense period
of matches (12). During this phase there may also be an
additional focus on technical and tactical preparation as opposed
to intense physical conditioning (13).This is commonly referred
to as the taper, which involves reducing the training loads
from a previously intense program to optimise recovery and
maximise performance (14,15). Athletes commonly employ
supplementation methods to enhance any adaptations from
training routines. Beta-alanine is an amino acid that is ingested
over several weeks to increase muscle carnosine content (16)
and improve exercise capacity and performance (17). It is
considered an effective ergogenic aid by the International
Olympic Committee (18), although the effects of beta-alanine
on football-specific protocols is unclear and contradictory.
Beta-alanine has previously improved YoYo IR2 performance
in amateur male footballers throughout a competitive season
(19), although YoYo IR1 was not improved in young elite
male basketball players (20). Evidence to support beta-alanine
supplementation during shorter-duration repeated sprints is
distinctly lacking (21–23) although supplementation alongside
plyometric training did lead to greater improvements in RAST
than training alone in female soccer players (11). This suggests
that the combination of training and beta-alanine may be
additive, something previously demonstrated with cycling sprint
training (24), although no study to date has investigated the
combined effect of a football-specific training period alongside
beta-alanine supplementation on football-specific performance
in females.
The aim of this study was to investigate the effect of beta-
alanine supplementation throughout a short-duration intense
football-specific training period prior to an international
competition on measures of high-intensity running performance.
We hypothesised that supplementation would lead to greater
improvements in exercise measures than any seen with
training alone.
METHODS
Participants
Twenty-four elite international under-20 (U20) female
footballers (age 18 ±1 y, height 1.67 ±0.07 m, body mass
62.7 ±7.4 kg) from different clubs competing in the elite
divisions of the Brazilian football pyramid that were part of the
national Brazilian team preparing for the South American U20
Women’s Championship, volunteered for the study and were
randomly assigned to receive either beta-alanine (BA, N=12)
or placebo (PL, N=12). Subjects had not taken any creatine
supplement in the 3 months prior to the study and had not taken
BA for at least 6 months. None of the subjects were vegetarian
and, therefore, would have encountered small amounts of
beta-alanine in their diet from the hydrolysis of carnosine and
its methyl derivatives in meat. The study was approved by the
institution’s Ethical Advisory Committee.
Experimental Design
All athletes in this study routinely performed the exercise
protocols as part of standard fitness testing throughout their
respective seasons. Participants performed the YoYo IR1, RAST,
and 20-m sprint test on two separate occasions, separated by
3 weeks of training and supplementation. The exercise tests
were performed in a standardized order: Sprint Test and the
YoYo IR1 in the morning and the RAST test in the evening.
All players performed the same 3-week standardized training
program, which consisted of 1 to 2 training sessions per day and
received 5 meals per day at standardized timepoints. Sleeping
and waking times during the training period were controlled and
identical for all athletes. Training and diet, including caffeine
consumption, in the 24 h period prior to the first main exercise
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Ribeiro et al. Short-Duration Beta-Alanine Supplementation Female Footballers
session were standardized and the athletes repeated this prior to
the second main session.
Supplement group allocation was conducted in blocks with
groups being equalized according to performance in the
YoYo IR1. Throughout the same 3-week period, participants
were supplemented with either 6.4 g·day−1of beta-alanine
(CarnoSynTM, NAI, USA) or placebo (maltodextrin; NAI, USA)
in sustained-release tablets. The dosing regimen consisted of
two 800 mg BA or PL tablets ingested four times per day (7
AM/12 PM/5 PM/10 PM). Participants ingested the supplements
alongside their standardized meals and a final dose before
bed, ensuring all had 100% compliance to the supplementation
regimen. No participant in either group reported any symptoms
of paraesthesia throughout supplementation.
Experimental Procedures
YoYo Intermittent Recovery Test – Level 1
The YoYo IR1 consists of repeated 2 ×20 m runs between
markers at progressively increasing speeds dictated by an audio
signal. At the end of each 2 ×20 m bout, individuals performed
10 s of active recovery between consisting of a 10 m (2 ×5 m)
walk. The test ended if the player failed to reach the finish line
within the given time frame on two consecutive occasions or if
the player felt unable to continue (volitional exhaustion). The
total distance covered (m) during the test was recorded as the
outcome measure.
Running Anaerobic Sprint Test (RAST) and 20-m
sprint
The RAST consisted of seven 20-m maximal sprints with a
passive 10 s recovery period between each sprint; the start of each
sprint was indicated by a beep from the photocell equipment
(CEFISE standard photocells, Brazil) which measured run time
for every individual sprint. The photocells were connected to
a computer with specific software (CEFISE, Brasil) for speed
analysis. Outcome measures were sprint time of each sprint
(s), total sprint time (s), mean, maximum and minimum
power output (W) [calculated as Power =(Body Mass ×
Distance2)/Time3] and fatigue index (FI, %) [calculated as FI
=(peak power - minimum power/peak power) ×100)] (10).
Participants also performed 3 separate attempts of a maximum
20 m sprint test, with 5 min passive recovery between efforts. To
start the sprint, the volunteer was positioned 1 m behind the
first photocell to prevent premature activation of the timer. The
timing of the start of each maximal sprint was determined by
the athlete.
Data Analysis
Data were analysed using the SAS statistical package (SAS R

University Edition, SAS Institute Inc., USA), and are presented
as mean ±1SD unless stated. Exercise data were analysed using
mixed model analysis with individuals assumed as a random
factor and supplementation (2 levels; BA and PL) and time (2
levels; Day 0 and 20) assumed as fixed factors. Repeated sprints
during the RAST were analysed using mixed model analysis with
individuals assumed as a random factor and supplementation
(2 levels; BA and PL), time (2 levels; Day 0 and 20) and sprint
number (7 levels; 0-7) assumed as fixed factors. Tukey–Kramer
FIGURE 1 | Distance covered during the YoYo IR 1 test in the beta-alanine
(BA) and placebo (PL) groups pre- and post-supplementation. *P=0.004.
Effect of time.
adjustments were performed when a significant F value was
obtained, and the significance level was set a priori at P≤0.05.
Individual responses for the YoYo IR1 were calculated according
to time-to-completion using the spreadsheet of Swinton et al. (25)
using 90% confidence intervals, a typical error calculated from
reproducibility data (26) and a smallest worthwhile change of 0.2
×the standard deviation of the control session (27). Due to issues
unrelated to the intervention (due to minor illness or injury, the
coaches instructed the athletes not to complete all protocols as
a precautionary measure), complete data for the YoYo IR1 was
obtained for 20 athletes (BA =10, PL =10) and 22 athletes
completed the 20-m sprint (BA =11, PL =11); all athletes
completed the RAST pre- and post-supplementation.
RESULTS
YoYo IR1
YoYo performance was not significantly different between groups
at baseline (BA: 644 ±114 m, PL: 513 ±125 m; P=0.07),
although this almost reached statistical significance. This might
be due to missing data (2 individuals from BA and 2 from
PL). There was a main effect of group (P=0.046), with lower
overall values in the PL vs. BA group, and time (P=0.004);
distance covered was lower post- versus per-supplementation
(−7.4 ±14.4%). The group ×time interaction did not reach
statistical significance (P=0.07; Figure 1). Individual data
analysis revealed that no individuals in either group improved
performance above the smallest worthwhile change during
the YoYo IR1, although two athletes in BA and one in PL
worsened performance.
RAST and 20 m Sprint
There was no effect of group (P=0.67) or time (P=0.45) for
sprint times during the RAST, but there was an effect of sprint
number (P<0.0001), reflecting an increase in time to complete
each sprint with increasing sprint number (Table 1). There were
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Ribeiro et al. Short-Duration Beta-Alanine Supplementation Female Footballers
TABLE 1 | Sprint times (s) during the RAST for the beta-alanine (BA) and placebo
(PL) groups pre- and post-supplementation.
BA PL
Pre Post Pre Post
Sprint 1 (s) 3.6 ±0.1 3.5 ±0.1 3.5 ±0.1 3.6 ±0.1
Sprint 2 (s) 3.6 ±0.2 3.6 ±0.1 3.6 ±0.1 3.6 ±0.1
Sprint 3 (s) 3.7 ±0.1* 3.7 ±0.2* 3.6 ±0.1* 3.7 ±0.2*
Sprint 4 (s) 3.8 ±0.2* 3.8 ±0.2* 3.7 ±0.1* 3.7 ±0.2*
Sprint 5 (s) 3.8 ±0.1* 3.8 ±0.2* 3.8 ±0.1* 3.8 ±0.2*
Sprint 6 (s) 3.9 ±0.2* 3.9 ±0.2* 3.8 ±0.1* 3.9 ±0.2*
Sprint 7 (s) 3.9 ±0.1* 3.9 ±0.2* 3.9 ±0.1* 3.9 ±0.1*
Total sprint time (s) 26.1 ±1.0 26.2 ±1.0 26.0 ±0.6 26.0 ±1.0
20-m sprint 3.5 ±0.1 3.5 ±0.2 3.5 ±0.1 3.4 ±0.1
P<0.0001 Effect of sprint, *indicates a significant difference from Sprint 1.
no group ×time ×sprint interaction effects for sprint times (P
=0.96). There were no group, time or group ×time interactions
for total time, maximum, mean and minimum power and fatigue
index during the RAST (all P>0.05) (Figure 2). There was no
effect of group (P=0.91), time (P=0.50) or group ×time
interaction (P=0.25) for the 20-m sprint test.
DISCUSSION
This study aimed to investigate the effect of BA supplementation
in high-level Brazilian female soccer players during a three-
week preparatory training period. The main findings showed that
responses to BA supplementation were not different from those
obtained with placebo and were unable to avoid decreases in
performance during the YoYo IR1, which likely occurred due to
high workloads imposed in this preparatory period. There were
no changes in repeated or maximal sprint performance.
The training load employed with these athletes resulted in a
reduced exercise capacity during the YoYo IR1 (−7.4%). Prior to
major events, such as an international tournament, it is common
to taper, namely reducing the training load from a previously
intense program in order to optimise gains and recovery and
maximise subsequent performance (14,15). Our data suggest
that, not only was the training intervention too intense in nature
to illicit improvements in exercise capacity, it actually worsened
performance which is contrary to the aims of the training.
Previous data from an under-20 female football team preparing
for the World Cup showed a progressive improvement in YoYo
IR1 performance leading up to competition (Tunstall H, personal
communication in (6)), although the authors suggest this was
reflective of a more focused fitness training schedule and the low
starting fitness levels of these female players. In the current study,
our athletes all plied their trade for top-level national sides, and
most were regular starters for their respective teams. It is possible
that a long grueling season took its toll on the players, and that
performance was a result of accumulated fatigue over the season
and would have reduced over this three-week period regardless
of the intense training. In fact, YoYo IR1 performance of these
FIGURE 2 | Sprint performance during the RAST for the beta-alanine (A) and
placebo (B) groups pre- (clear bars) and post- (striped bars) supplementation.
*P<0.0001. Effect of sprint number.
athletes prior to the training and supplementation intervention
was lower than that shown previously in elite female footballers
(7,9), which provides support for this theory.
Short-term BA supplementation (3 weeks) was unable
to attenuate this training-associated decline in YoYo IR1
performance. The lack of an effect shown here is line with
previous research showing no changes in YoYo IR1 in young elite
male basketball players with BA supplementation (20), although
BA did improve YoYo IR2 (19) in amateur male footballers. The
YoYo IR2 is initiated at a higher intensity than the YoYo IR1,
with a higher contribution from anaerobic glycolytic pathways
increasing the contribution of buffering capacity to performance
(9), making it more susceptible to improvements with BA. It is
also possible that the relatively short loading period in this study
did not meet the threshold necessary for a sufficient increase
in muscle carnosine to elicit performance improvements. Smith
et al. (28) showed similar improvement in cycling capacity
following 3 weeks of high-intensity interval training with both
BA and PL, but greater improvements from weeks 3–6 were
shown with BA. It is currently unknown what the minimal
necessary increase in muscle carnosine is to elicit a performance
improvement (29) and any definitive conclusions here are not
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Ribeiro et al. Short-Duration Beta-Alanine Supplementation Female Footballers
possible due to the lack of muscle carnosine content analysis in
the current study. Had the athletes commenced supplementation
prior to the training phase, thus ensuring increased muscle
carnosine content prior to the intense training period, it is
possible that results may have been different. However, we were
unable to enforce the supplementation protocol prior to the
international period during which we had access to the players,
a potential consequence of working with elite club players on
international duty. As it stands, short-term BA supplementation
was unable to attenuate the decline in YoYo IR1 performance
following the training period in this study.
Neither training nor supplementation led to changes in
repeated sprint ability or sprint performance. These data are
in line with previous studies showing no effect of BA on
short-duration repeated sprints in team sports players (21,
22). However, previous research in female football players
has shown BA supplementation to improve mean power
output during repeated 30-s Wingate sprints (30) and induce
greater improvements in repeated-sprint tests when combined
with plyometric training compared to training alone (11).
It is possible that the highly trained nature of our athletes
contributed to these results, since meta-analytical data has
shown well-trained individuals to achieve smaller performance
gains with supplementation than non-trained individuals (17).
The aforementioned studies recruited university level (30) and
amateur (11) players, while we employed elite youth players. It is
also important to again emphasise that the lack of any changes
in these tests may similarly be due to the intense nature of the
training program, inhibiting any potential adaptations with or
without supplementation.
One of the strengths of this study is that it was a real-
world intervention in which we implemented a double-blind
placebo-controlled supplementation protocol in top-level female
athletes who were part of a competitive international set-up
performing their normal pre-competition training program. The
standardized pre-tournament training camp provided a unique
environment that required all athletes to undergo identical daily
routines such as training, nutritional intake and sleep, thus
removing several variables which could contribute to individual
variability. Indeed, our data showed striking consistency with all
but one individual in BA showing a reduction in distance covered
during the YoYo IR1, while six athletes in PL also covered less
distance; however, statistical analysis revealed that only two in BA
and one in PL could be considered to have worsened performance
with >90% certainty (25). The controlled nature of this study has
great practical applicability to similar athletes undergoing these
types of intervention by showing BA to be ineffective during such
a short and intense training period. However, alternative methods
might be implemented, such as lighter training loads and earlier
implementation of the supplementation regime, which might
lead to different results. Indeed, this study also highlights the
delicate nature of working with an international team since we
could not make any changes to their usual routines until their
individual seasons with their respective national clubs had ended.
PRACTICAL APPLICATIONS
Intense preparatory training periods prior to international
competitions may place unnecessary strain on top-level
footballers following a grueling season. Our data suggest
that international teams may inadvertently overload
their players leading to a reduced high-intensity exercise
capacity in these players. Reduced exercise capacity was
not counteracted by short-duration BA supplementation,
although it is possible that the supplementation protocol
was sub-optimal. Perhaps more communication between
clubs and international teams may facilitate this process and
avoid overreaching or overtraining, while prior initiation
of the supplementation protocol would further benefit any
potential adaptation.
CONCLUSIONS
Top-level female footballers involved in this intense 3-week
training period prior to a competition worsened their high-
intensity intermittent exercise capacity, and this negative result
was not attenuated by a short-duration BA supplementation
protocol throughout the same period. Further work is necessary
to elucidate whether adapted training protocols and BA dosing
regimens could lead to better results.
DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to
the corresponding author.
ETHICS STATEMENT
The studies involving human participants were reviewed and
approved by University of São Paulo. The patients/participants
provided their written informed consent to participate in
this study.
AUTHOR CONTRIBUTIONS
GR, AR, EP, VC, MB, PG, BG, and BS contributed to the
conception and design of the study. RR, EP, and BS organized the
database and performed the statistical analysis. RR, BD, AG, EP,
and BS wrote the first draft of the manuscript. GR, AR, VC, MB,
PG, and BG contributed to the subsequent versions. All authors
contributed to manuscript revision, and read and approved the
final submitted version.
FUNDING
No specific funding was received for this investigation. BD
(2019/06140-5) and BS (2016/50438-0 and 2017/04973-4) have
been financially supported by Fundação de Amparo à Pesquisa
do Estado de São Paulo.
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Ribeiro et al. Short-Duration Beta-Alanine Supplementation Female Footballers
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Conflict of Interest: BS has previously received financial support from Natural
Alternatives International (NAI), a company that produces BA, to undertake a
study unrelated to this one. NAI has also provided BA supplements free of charge
for this and further experimental investigations and supported open access page
charges for numerous publications involving the authors. NAI have not had any
input (financial, intellectual, or otherwise) into this original investigation.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
Copyright © 2020 Ribeiro, Duarte, Guedes da Silva, Ramos, Rossi Picanço, Penna,
Coswig, Barbalho, Gentil, Gualano and Saunders. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY).
The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these
terms.
Frontiers in Nutrition | www.frontiersin.org 6April 2020 | Volume 7 | Article 43