**1. 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

48% of athletes use nutritional supplements and ergogenic aids [3], claiming that certain components, such as creatine, caffeine, sodium bicarbonate, and BA, contribute to an improvement in their physical performance [5–7].

Specifically, BA is a non-essential amino acid synthesized in the liver and found in products of animal origin [8]. Evidence shows that poultry, beef, and fish are products with a large BA content [9]. BA has been consistently shown to increase levels of carnosine (CA) in human skeletal muscle [9–12]. This last substance is synthesized by CA synthase when bonding BA with L-histidine [13]; CA is found in the muscular tissue and acts as a buffer of hydrogen protons (H+) in high-intensity physical exercises of short duration [11,14]. This is why athletes who follow a vegetarian diet will have lower muscular CA concentrations than those who follow an omnivorous diet [15].

When performing high-intensity exercises, due to the predominant energetic system (anaerobic metabolism of carbohydrates), a high release of H<sup>+</sup> takes place, which leads to a decrease in pH [12]. This pH decrease can negatively affect the metabolic processes of phosphocreatine resynthesis, inhibit contractile processes, and diminish the glycolytic rate—all these factors contribute to the onset of muscular fatigue [14]. Some studies have concluded that an elevated muscular CA concentration could buffer between 8–15% of H+, opening the possibility of maximizing physical effort for a longer period of time [1]. On the other hand, other studies have shown that CA and L-histidine supplementation do not increase the bioavailability of intramuscular CA [5,14]. For this reason, and considering BA as a precursor in CA formation, several studies have shown an increase between 40–80% of intramuscular CA post BA supplementation [1,9–11,16]. In this regard, the acute effect of BA supplementation has been tested in doses of 30 mg·kg−<sup>1</sup> of body mass and prolonged supplementation with doses ranging from 2.0 to 6.4 g/day for periods of time between 4 and 10 weeks [12,17]. At the same time, BA can be found as the main ingredient in multi-ingredient pre-workout supplements, although it is worth mentioning that these products have a lower dosage than that studied clinically [18]. Specifically, lower pH values have been measured after 4 min of high-intensity exercise [19] and the drop of pH is one of the factors responsible for the increase in ventilatory responses [20]. In parallel, the background shows that BA supplementation reported only one secondary effect, paresthesia [21,22]; this is a sensation of flushing associated with an irritant tingling in the ears, scalp, hands, and torso [23].

Related to pH stabilization, there are several studies that have used ergogenic aids to improve physical performance in aerobic–anaerobic transition zones [8,24]. The aerobic–anaerobic transition zone corresponds to an intensity range between aerobic threshold and anaerobic threshold [25] and may serve as a basis for assessing endurance performance individually as well as for prescribing intensities in endurance training [26]. In this regard, BA is among the ergogenic aids used to increase performance in aerobic–anaerobic transition zones [8,9,27]. One study has evaluated the effect of BA supplementation on physical performance, showing an improvement of 13.9% in ventilatory threshold [20]. In addition, another study reported that BA supplementation for 28 days enhanced sub-maximal endurance performance by delaying the onset of blood lactate accumulation (OBLA) [8]. However, other investigations have not found significant results in athletic performance [12], specifically in rowers [28], and trained cyclists [29] with BA supplementation.

The existing evidence shows controversial results that make it impossible to categorize or ensure that BA supplementation improves physical performance in aerobic–anaerobic transition zones (performance mainly connected to ventilator parameters). Hence, the primary aim of this systematic review and meta-analysis was to analyze the effects of BA supplementation on physical performance in aerobic–anaerobic transition zones. Likewise, the effects of different doses and supplementation times with BA were identified.

#### **2. Materials and Methods**

#### *2.1. 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.

#### *2.2. 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% VO2max), (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.

#### *2.3. 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].

#### *2.4. Outcome Measures*

The articles were examined regarding the effect of BA supplementation on physical performance in aerobic–anaerobic transition zones (60–100% VO2max) [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% VO2max), 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 VO2max) [33,34], while the minimum lower limit in the aerobic–anaerobic transition zone was 60% of VO2max [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 VO2max (LO2·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.
