Effects of Beta-Alanine Supplementation on Physical Performance in Aerobic–Anaerobic Transition Zones: A Systematic Review and Meta-Analysis

Beta-alanine supplementation (BA) has a positive impact on physical performance. However, evidence showing a benefit of this amino acid in aerobic–anaerobic transition zones is scarce and the results controversial. The aim of this systematic review and meta-analysis is to analyze the effects of BA supplementation on physical performance in aerobic–anaerobic transition zones. At the same time, the effect of different dosages and durations of BA supplementation were identified. The search was designed in accordance with the PRISMA® guidelines for systematic reviews and meta-analyses and performed in Web of Science (WOS), Scopus, SPORTDiscus, PubMed, and MEDLINE between 2010 and 2020. The methodological quality and risk of bias were evaluated with the Cochrane Collaboration tool. The main variables were the Time Trial Test (TTT) and Time to Exhaustion (TTE) tests, the latter separated into the Limited Time Test (LTT) and Limited Distance Test (LDT). The analysis was carried out with a pooled standardized mean difference (SMD) through Hedges’ g test (95% CI). Nineteen studies were included in the systematic review and meta-analysis, revealing a small effect for time in the TTT (SMD, −0.36; 95% CI, −0.87–0.16; I2 = 59%; p = 0.010), a small effect for LTT (SMD, 0.25; 95% CI, −0.01–0.51; I2 = 0%; p = 0.53), and a large effect for LDT (SMD, 4.27; 95% CI, −0.25–8.79; I2 = 94%; p = 0.00001). BA supplementation showed small effects on physical performance in aerobic–anaerobic transition zones. Evidence on acute supplementation is scarce (one study); therefore, exploration of acute supplementation with different dosages and formats on physical performance in aerobic–anaerobic transition zones is needed.


Introduction
A proper diet is one of the main factors in the improvement of physical performance. However, sometimes it is not enough to meet the energetic demands of training sessions [1]. For this reason and with the aim of maximizing physical performance, the use of nutritional supplements is widespread in sport [2], even more in younger athletes [3]. Nutritional supplements, such as protein and carbohydrates, are concentrated nutrient sources that substitute or complement the use of certain foods, while ergogenic aids, such as caffeine, creatine, or beta-alanine (BA), are pharmacological agents used with the aim of enhancing physical performance [4]. In this regard, one study showed that

Literature Search Strategies
In order to perform this review, a thorough electronic search was carried out in several databases and search engines. Articles published in Web of Science (WOS), Scopus, SPORTDiscus, PubMed, and MEDLINE were included. A search limit was established from January 2010 to February 2020.
The bibliographic search was performed in accordance with the PRISMA ® statement guidelines for systematic reviews and meta-analyses [30]. In each of the aforementioned databases, the search included hits in the title, abstract, and key words search fields. The following key words were combined with Boolean operators AND/OR: [("b-alanine" OR "beta-alanine" OR "b-alanine supplementation" OR "beta-alanine supplementation") AND ("maximal aerobic speed" OR "maximal oxygen uptake" OR "maximal aerobic consumption" OR "endurance")]. One of the authors performed the search, and two reviewed the studies. Together, they decided whether the studies were appropriate for inclusion.

Inclusion and Exclusion Criteria
The importance of each study was assessed according to the following inclusion criteria: (1) BA supplementation, either acute or chronic supplementation, (2) experimental design studies, (3) healthy adults, (4) studies that included physical performance evaluation in the aerobic-anaerobic transition zone (60-100% VO 2 max), (5) studies that included Time Trial Tests (TTT), or Time to Exhaustion (TTE) tests for physical performance evaluation, (6) studies that stated a baseline and control group, (7) studies showing negative and positive changes in TTT or TTE tests, and (8) studies published in English and Spanish. The studies that failed to fulfill the inclusion criteria were not considered in the systematic review nor the meta-analysis. Possible discrepancies were resolved through discussion until a consensus was reached.

Chronic and Acute Supplementation
Regarding the classification of the supplementation protocols assessed in this systematic review, acute supplementation was considered to be the one in which a unique dose of BA was used between 0 min and 24 h prior to physical exercise, while chronic supplementation was considered those protocols that used repeated dosages of BA for more than one day and up to 10 weeks [31].

Outcome Measures
The articles were examined regarding the effect of BA supplementation on physical performance in aerobic-anaerobic transition zones (60-100% VO 2 max) [26,32]. The primary outcome used for the systematic review and meta-analysis were (a) TTT and (b) TTE tests (Limited Time Test (LTT) and Limited Distance Test (LDT)). In order to establish the upper limit in the aerobic-anaerobic transition zone (100% VO 2 max), the minimum time used on the TTT and TTE test (LTT) was 300 s (the literature sets this as the minimum amount of time needed to determine VO 2 max) [33,34], while the minimum lower limit in the aerobic-anaerobic transition zone was 60% of VO 2 max [32]. These limits were set in order to include studies showing results of 5-63 min [21,35]. The systematic review and meta-analysis also included secondary outcomes stated in the studies. These secondary variables were (a) capillary lactate (mmol·L −1 ), (b) absolute VO 2 max (LO 2 ·min −1 ), (c) HR (bpm), and (d) ratings of perceived exertion (RPE) according to the Borg scale [36]. It is important to mention that studies were excluded from the systematic review and meta-analysis if they only showed secondary results in the in extenso reading. Median values, standard deviations (SD), and sample sizes were included for the statistical analysis of the meta-analysis, for both the primary and secondary outcomes. If the selected studies did not include numerical data, it was requested of the authors, or if the data were plotted as figures, the values were estimated based on the pixel count. Additionally, the studies that declared paresthesia symptoms in their subjects were also included.

Publication Bias
Publication bias was assessed using Egger's statistical test. This test determined the presence of bias at p ≤ 0.05 [37]. Funnel plots were created to interpret the general effect, followed by an Egger's statistic to confirm or refute publication bias. Egger's analysis suggested that the primary variables did not show publication bias: (a) TTT: z = 1.35, p = 0.18; (b) LTT: z = 1.90, p = 0.06; (c) LDT: z = 1.85, p = 0.06 ( Figure 1). figures, the values were estimated based on the pixel count. Additionally, the studies that declared paresthesia symptoms in their subjects were also included.

Publication Bias
Publication bias was assessed using Egger's statistical test. This test determined the presence of bias at p ≤ 0.05 [37]. Funnel plots were created to interpret the general effect, followed by an Egger's statistic to confirm or refute publication bias. Egger's analysis suggested that the primary variables did not show publication bias: (a) TTT: z = 1.35, p = 0.18; (b) LTT: z = 1.90, p = 0.06; (c) LDT: z = 1.85, p = 0.06 (Figure 1).

Quality Assessment of the Experiments
The methodological quality and risk of bias for each selected study were assessed through a Cochrane Collaboration guideline [38]. The list was divided into six different domains: selection bias (random sequence generation, allocation concealment), performance bias (blinding of participants and personnel), detection bias (blinding of outcome assessment), attrition bias (incomplete outcome data), reporting bias (selective reporting), and other types of bias (declaration of conflict of interest). For each item, the answer to a question was considered; when the question was answered with a "Yes", the bias was low; when it was "No", the bias was high; when it was "Unclear", the possible bias was connected to a lack of information or uncertainty. The full details of each study and domains are presented in Figures 2 and 3.

Quality Assessment of the Experiments
The methodological quality and risk of bias for each selected study were assessed through a Cochrane Collaboration guideline [38]. The list was divided into six different domains: selection bias (random sequence generation, allocation concealment), performance bias (blinding of participants and personnel), detection bias (blinding of outcome assessment), attrition bias (incomplete outcome data), reporting bias (selective reporting), and other types of bias (declaration of conflict of interest). For each item, the answer to a question was considered; when the question was answered with a "Yes", the bias was low; when it was "No", the bias was high; when it was "Unclear", the possible bias was connected to a lack of information or uncertainty. The full details of each study and domains are presented in

Statistical Analysis
In order to evaluate the quality of the experiments and interpret the risk of bias values, Review Manager version 5.4 was used (Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2014). The same software was used to perform a descriptive and statistical analysis of the meta-analysis. To compare the supplementation of BA versus the placebo (PL), the number of participants, standardized mean difference (SMD), and standard error of SMD were analyzed for each study. Hedges' g test was used to calculate the SMD of each study [39]. The overall effect and its 95% confidence interval (CI) were calculated by weighting the SMD by the inverse of the variance. Additionally, the SMD of both the BA supplemented and PL groups were subtracted to obtain the net effect size (ES), which was used together with the pooled SD of change to calculate the variance (ES = [mean BA − mean PL]/SD); to interpret the magnitude of the ES, Cohen's criteria were followed: <0.2, trivial; 0.2-0.5, small; 0.5-0.8, moderate; and >0.8, large [40].
The I 2 statistic was calculated as an indicator of the percentage of observed total variation within studies due to real heterogeneity rather than chance. I 2 values are included from 0 to 100%, representing a small amount of inconsistency between 25% and 50%, a medium amount of heterogeneity between 50% and 75%, and a large amount of heterogeneity when the I 2 value was higher than 75%. In this sense, low, moderate, and high adjectives would be accepted referring to I 2 values of 25%, 50%, and 75%, respectively, although a restrictive categorization would not be adequate in all circumstances [41].

Main Search
The literature search through electronic databases identified 323 articles of which 177 were duplicates. The remaining 146 articles were filtered by title and abstract, and 57 studies remained to be read and analyzed. After a review of those 57 studies, 40 were eliminated because they did not meet the inclusion criteria. In the search for articles oriented by bibliographic references, two extra studies were added. As a result, 19 articles were included for the systematic review and meta-analysis. The search strategy and study selection are shown in Figure 4. Out of 19 studies, eight considered time in a TTT to assess the effect of BA supplementation on physical performance [29,[42][43][44][45][46][47][48], nine used time (LTT) on a TTE test to assess the same effect [5,21,22,35,[49][50][51][52][53], one considered the distance (LDT) on a TTE test [28], while one considered both the time (LTT) and the distance (LDT) on a TTE test [17] ( Table 1).

Effect of BA on Time Trial Tests
Eight studies were considered for this analysis [29,[42][43][44][45][46][47][48]. However, two of them included two TTTs in the research design [29,44]. For the meta-analysis, the study by Bellinger et al. [29] was considered as two independent designs (TTT of 4 and 10 km on a cycle ergometer, respectively). In the same way, the study by Bellinger et al. [44] was considered as two independent designs (TTT of 4 and 10 km on a cycle ergometer, respectively). Thus, 10 studies were included in the meta-analysis that calculated the effect of BA supplementation on time in TTT. Figure 5 shows that BA supplementation generates a small and non-significant effect on physical performance in TTT (SMD, −0.36; 95% CI −0.87-0.16; p = 0.18). The meta-analysis showed moderate heterogeneity among the studies reviewed (I 2 = 59%; p = 0.01). Out of the 10 studies analyzed, seven of them declared a beneficial effect of supplementation with BA on physical performance in TTT [29,42,43,[46][47][48]. Out of these studies, the research of Santana et al. [48] presented a large ES (−6.70). On the other hand, three of the 10 studies showed a neutral or prejudicial effect after BA supplementation [44,45].

Effect of BA on the Limited Time Test
Ten studies were considered for this analysis [5,17,21,22,35,[49][50][51][52][53]. However, one of them included two experimental groups for the LTT in their research design [21]. For the meta-analysis, two experimental groups presented by Smith-Ryan et al. [21] were considered as two independent studies (LTT at 90% of VO 2 max on a treadmill for women and LTT at 90% of VO 2 max on a treadmill for men). This way, 11 studies were included in the meta-analysis that calculated the effect of BA supplementation on the TTE test. Figure 6 shows that BA supplementation generated a small and non-significant effect for time on the TTE test (SMD, 0.25; 95% CI −0.01-0.51; p = 0.06). A meta-analysis showed low heterogeneity among the reviewed studies (I 2 = 0%; p = 0.53). Out of the 11 studied and analyzed, eight showed a positive effect of BA on time in the LTT [5,17,21,22,[49][50][51][52]. Out of these studies, Furst et al. [49] showed a large ES (1.64). On the other hand, three of the 11 studies showed an unbeneficial effect after BA supplementation [21,35,53].    supplementation generates a small and non-significant effect on physical performance in TTT (SMD, −0.36; 95% CI −0.87-0.16; p = 0.18). The meta-analysis showed moderate heterogeneity among the studies reviewed (I 2 = 59%; p = 0.01). Out of the 10 studies analyzed, seven of them declared a beneficial effect of supplementation with BA on physical performance in TTT [29,42,43,[46][47][48]. Out of these studies, the research of Santana et al. [48] presented a large ES (−6.70). On the other hand, three of the 10 studies showed a neutral or prejudicial effect after BA supplementation [44,45].

Effect of BA on the Limited Time Test
Ten studies were considered for this analysis [5,17,21,22,35,[49][50][51][52][53]. However, one of them included two experimental groups for the LTT in their research design [21]. For the meta-analysis, two experimental groups presented by Smith-Ryan et al. [21] were considered as two independent studies (LTT at 90% of VO2max on a treadmill for women and LTT at 90% of VO2max on a treadmill for men). This way, 11 studies were included in the meta-analysis that calculated the effect of BA supplementation on the TTE test. Figure 6 shows that BA supplementation generated a small and non-significant effect for time on the TTE test (SMD, 0.25; 95% CI −0.01-0.51; p = 0.06). A meta-analysis showed low heterogeneity among the reviewed studies (I 2 = 0%; p = 0.53). Out of the 11 studied and analyzed, eight showed a positive effect of BA on time in the LTT [5,17,21,22,[49][50][51][52]. Out of these studies, Furst et al. [49] showed a large ES (1.64). On the other hand, three of the 11 studies showed an unbeneficial effect after BA supplementation [21,35,53].

Effect of BA on the Limited Distance Test.
Two studies were considered for this analysis [17,28]. However, the study of Baesley et al. [28] included two experimental groups for the LDT in their research design (30 min on a rowing ergometer with 2.4 g/day of BA supplementation every 24 h and 30 min on a rowing ergometer with 4.8 g/day of BA supplementation every 48 h). In this way, three studies were included in the metaanalysis that calculated the effect of BA supplementation on the TTE test. Figure 7 shows that BA supplementation generates a large and non-significant effect on distance in the TTE test (SMD, 4.27; 95% CI −0. 25-8.79; p = 0.06). The meta-analysis showed high heterogeneity among the studies reviewed (I 2 = 94%; p = 0.00001). All studies analyzed declared a beneficial effect of supplementation with BA on physical performance in LDT [17,28].

Effect of BA on the Limited Distance Test
Two studies were considered for this analysis [17,28]. However, the study of Baesley et al. [28] included two experimental groups for the LDT in their research design (30 min on a rowing ergometer with 2.4 g/day of BA supplementation every 24 h and 30 min on a rowing ergometer with 4.8 g/day of BA supplementation every 48 h). In this way, three studies were included in the meta-analysis that calculated the effect of BA supplementation on the TTE test. Figure 7 shows that BA supplementation generates a large and non-significant effect on distance in the TTE test (SMD, 4.27; 95% CI −0.25-8.79; p = 0.06). The meta-analysis showed high heterogeneity among the studies reviewed (I 2 = 94%; p = 0.00001). All studies analyzed declared a beneficial effect of supplementation with BA on physical performance in LDT [17,28].
4.8 g/day of BA supplementation every 48 h). In this way, three studies were included in the metaanalysis that calculated the effect of BA supplementation on the TTE test. Figure 7 shows that BA supplementation generates a large and non-significant effect on distance in the TTE test (SMD, 4.27; 95% CI −0. 25-8.79; p = 0.06). The meta-analysis showed high heterogeneity among the studies reviewed (I 2 = 94%; p = 0.00001). All studies analyzed declared a beneficial effect of supplementation with BA on physical performance in LDT [17,28].

Effect of BA Supplementation on Secondary Outcomes
Of the total of 19 studies included in the systematic review and meta-analysis, 17 of them reported on different parameters of physical performance. These parameters were defined as secondary outcomes and included blood lactate concentration ([La]), VO2max, RPE, and HR [32].

Effect of BA Supplementation on Secondary Outcomes
Of the total of 19 studies included in the systematic review and meta-analysis, 17 of them reported on different parameters of physical performance. These parameters were defined as secondary outcomes and included blood lactate concentration ([La]), VO 2 max, RPE, and HR [32].

Paresthesia
At the end of this review, out of the 19 studies included in the systematic review and meta-analysis, four of them reported paresthesia [21,22,29,51] (Table 1).

Discussion
In connection with the studies included in the systematic review and meta-analysis, the results showed that BA supplementation presents an ES ranging from a small (0.2-0.5) to a large magnitude (>0.8) in aerobic-anaerobic transition zones. At the same time, the results showed that changes in physical performance are associated with both acute and chronic BA supplementation, while the administered doses ranged from 1.5−6.4 g/day in periods ranging from 1 h before physical tests (acute supplementation) to 10 weeks with one or several doses during the day (chronic supplementation).
At the end of this review, several studies concluded that the increase in physical performance after BA supplementation is due to an increase in muscular CA concentrations [21,42,53]. The ergogenic effect that generates increased CA is associated with intracellular regulation of pH (buffer), an increase in Calcium (Ca 2+ ) sensitivity in type I and II muscle fibers, and an increase in Ca 2+ /H + ion exchange; as a consequence, these events showed an increase in muscular contractility [1]. For this reason, direct supplementation with CA has been studied with inconclusive results [14,54,55], since CA is degraded into BA and L-histidine in the stomach [5]. Specifically, the low effectiveness of direct supplementation with CA is related to the fact that L-histidine has a larger presence in plasma than BA [1]. Because of this, BA supplementation shows better results than CA supplementation [44,50].
At the end of this review, the only secondary effect reported and associated with BA supplementation was paresthesia [21,22]. This is a sensation of flushing associated with an irritant tingling in the ears, scalp, hands, and torso [23]. The process responsible for paresthesia is the release of L-histidine to form CA [9,12,27]. Paresthesia is transitory and can be avoided by dosing and ingesting BA in smaller portions throughout the day [9,12,27].

Effect of BA on the Time Trial Test and Time to Exhaustion Test
BA supplementation and the subsequent increase in CA could diminish H + circulation and prevent the drop in intracellular pH during high-intensity exercise [50]. In fact, CA has been described as the main buffering substance of H + at the muscular level [56]. Previous studies have stated that blood and muscular acidosis limit muscular contractility, which would favor the onset of fatigue [17,29,47,50]. At the same time, due to an increase in Ca 2+ sensitivity to type I fibers, it has been mentioned that BA supplementation can improve muscular contractile properties, delaying fatigue onset [17,57].
As mentioned above, the performance increase in aerobic-anaerobic transition zones is associated with greater availability of muscular CA [20,50]. This way, evidence has shown that prolonged BA supplementation in doses ranging from 2.0-6.4 g/day for 4-10 weeks can increase CA concentrations between 64-80% [9]. In connection with acute supplementation in aerobic-anaerobic transition zones, evidence is scarce [17]. In this regard, Huerta et al. [17] performed supplementation with 30 mg·kg −1 body mass (1.5-2.1 g/day) of BA 60 min prior to a TTE test. These researchers obtained an average increase of 40.5 s at the end of the study (p < 0.05). Despite that, and due to the limited evidence relating acute supplementation with BA on physical performance in aerobic-anaerobic transition zones, it is impossible to guarantee a real effect in this physiological zone. However, the increase in physical performance observed in this review is supported by greater bioavailability of CA, an increase that is observed shortly after the intake of BA [58]. This raises the possibility of studying the acute effects of BA using different protocols and observing the real effects in aerobic-anaerobic transition zones.
The ES for distance on the TTE test was large (ES = 4.27), while TTT and time on the TTE test was small (ES = −0.36 and 0.25, respectively). In light of these results, these last values show a small effect of BA supplementation on physical performance in aerobic-anaerobic transition zones. However, considering that an elite athlete's performance is bound by extremely tight margins (probably difficult to measure statistically), in real practice, a small ES could be of great importance, since it has been proven that in world finals, differences lower than 3% can be found between first and last place [1].

Effect of BA on Secondary Outcomes
BA supplementation could prevent the drop in intracellular pH during high-intensity exercise (due to an increase in muscular CA bioavailability) and, as a consequence, generate less lactate accumulation with the same intensity of physical exercise [48,50]. Regarding lactate accumulation, it is important to mention that this is not the cause of H + accumulation, but a high intensity of exercise produces a decrease in pH and an increase in intramuscular and blood [La] simultaneously, transforming lactate in a good marker of physical effort [8]. Despite the theoretical background, the meta-analysis showed a trivial effect on [La] post effort (ES = 0.16).
The influence of BA supplementation on aerobic performance has been widely studied [14,20,27,59]; however, the meta-analysis showed a trivial effect of BA on VO 2 (ES = 0.17) [51,60]. Apparently, the increase in VO 2 is less dependent on the buffer qualities that BA supplementation produces [20]. It is possible that the improvement in VO 2 reported in some studies included in the meta-analysis is more connected to physical training in aerobic-anaerobic transition zones than to BA supplementation [61,62].
In connection to RPE, some studies have shown a good correlation between RPE and HR during physical exercise in healthy subjects (1 point of RPE equals approximately 10 bpm). More so, the metabolic thresholds have been associated with specific values on the Borg scale [36]. Likewise, it has been shown that a lower value of RPE for the same workload entails a metabolic adaptation after the training process [63]. Despite these lines of theoretical evidence, the studies included in the meta-analysis showed a trivial effect on RPE reported by the participants (ES = 0.03). This value can be derived from the level of demand experienced by the participant; it is also possible that they exerted themselves to the maximum effort in all tests, reaching the upper limits of the RPE scales used [36].
As a consequence improved cardiac contractility, it has been described that CA can increase HR [53]. In addition, intracellular pH has proven to be a modulator of cardiac function, increasing the entrance of Ca 2+ during action potentials, facilitating cardiac contraction [64]. This information makes it possible to anticipate an increase in HR after BA supplementation [53]. However, HR is dependent on the intensity of physical effort; hence, if the participants exerted themselves to the maximum in all tests, it is likely that post-effort HR values would not show major variations when supplementing with BA (ES = 0.30).
Finally, due to a limited number of studies, only the secondary outcomes mentioned above were used. Subdividing the 10 TTT studies and 11 TTL studies to perform a meta-analysis by gender, age, exercise modalities, or physical activity level would have generated a bias in the information obtained [38].

Limitations
The main limitations of this research were the access to information and unspecific data reported by some studies included in the systematic review and meta-analysis. However, the limitations were solved by contacting the authors of each study. Only one document was not included because no answer was received. Another important limitation in this review was the limited number of studies that used TDL as a primary outcome [17,28].

Conclusions
Both acute and chronic supplementation with BA in doses of 1.5-6.4 g/day showed a small and non-significant effect on physical performance in aerobic-anaerobic transition zones. Physiologically, this positive change is due to the buffer effects generated by the larger bioavailability of intracellular CA, which allows for a delay in the onset of fatigue in the TTT and TTE tests within this specific physiologic zone. That is why small changes in individual performance must be considered, since they can be the difference between success and failure among high-level and elite athletes.
Furthermore, the findings showed evidence that acute supplementation with BA is scarce, generating alternatives for researchers to study the effect of this form of supplementation with different BA doses and formats on performance in aerobic-anaerobic transition zones.

Practical Applications
Coaches and athletes looking for an ergogenic aid to enhance physical performance in aerobic-anaerobic transition zones should consider both acute and chronic supplementation with BA. The dosage can range from 30 mg·kg −1 of body mass in acute supplementation to 6.4 g/day in chronic supplementation. The latter may be administered in several doses per day. However, it is advisable to check the dosage and supplementation formats with qualified professionals.
Finally, in order to avoid the presence of paresthesia after supplementation with BA, it is recommended that BA be dosed and ingested in small portions throughout the day (the amount suggested for these doses is 1.6 g of BA per dose) [9]. The second recommendation to avoid paresthesia is to also ingest a large amount of carbohydrates 60 min before ingesting BA (the suggested carbohydrate load is 2 g·kg −1 of body mass) [17].