As persons exercise to voluntary failure, multiple mechanisms (psychological, neurological, metabolic, etc.) may undermine performance. Since carnosine is an intramuscular compound, it is important to examine conditions within cells as exercise progresses to the point of fatigue. In the quest for sufficient ATP, perhaps the greatest exercise-induced intracellular change is the rapid accrual of lactate and H⁺ that typifies metabolic acidosis (Figure 3). Lactate and acid metabolites accrue commensurate to the degree by which intracellular aerobic capacity is exceeded, such as when anaerobic glycolysis serves as the primary ATP resynthesis pathway [30]. At maximal exercise rates, such bouts last from 15 to 240 s [4,30]. Intramuscular glycolytic rates may rise 1000-fold as persons go from rest to supramaximal exercise [31].

With supramaximal exercise, intracellular ATP declines up to 40% and causes a near complete depletion of phosphocreatine as well as elevations in lactate and H⁺ [32]. Intramuscular pH, from resting values of ~7.1, may decline from supramaximal activity to less than 6.5, which represents a four-fold rise in H⁺ and an intracellular increase of up to 54 mmol·kg⁻¹ [33,34,35]. When such increases are added to other reactions that raise the total proton load intracellular H⁺ levels exceed 100 mmol·kg⁻¹ [36]. Concomitant lactate increases also occur with higher glycolytic rates; intramuscular and plasma levels for the metabolite rise by up to 40 and 25 mmol·L⁻¹ [34,36,37,38]. A strong linear relationship exists between muscle pH decrements and intracellular lactate and pyruvate values [39]. Thus examinations of blood lactate changes over time, which quantify the rate of the metabolite’s entry into and removal from the vasculature, foretell concurrent intracellular H⁺ changes in response to, and recovery from, exercise.

Upon cessation of exercise, force output and intracelluar pH restoration is rapid, yet the former recovers at a faster rate [32,33]. For each measure, recovery is slower than their rate of loss incurred from supramaximal exercise [32,33]. Adverse intracellular changes from H⁺ accrual include phosphofructokinase inhibition, impaired phosphocreatine resynthesis, slower cross bridge transitions from low- to high-force states, losses in maximal shortening velocity, lowered glycolytic rates, competitive inhibition with Ca²⁺ at the troponin C subunit, and delayed Ca²⁺ reuptake by the sarcoplasmic reticulum [3,30,40]. Such changes compromise exercise and recovery rates, which is a concern if successive bouts of supramaximal activity are to occur [32]. If contractions proceed at high force outputs, which are common to supramaximal exercise, high H⁺ levels also impair intramuscular Ca²⁺ release and compromise Ca²⁺-ATPase activity [41].

To cope with such conditions cells utilize multiple mechanisms to remove lactate and H⁺, as well as buffers to mitigate the effects of metabolic acidosis. Lactate and H⁺ efflux is facilitated by monocarboxylate transporters such as MCT1 and MCT4 [40]. Unlike carnosine, which appears to have no ceiling with respect to its intramuscular concentration [18], other intracellular buffers such as proteins, bicarbonates, citrates and NAD⁺-dependent redox reactions, are under tight homeostatic control [42]. In untrained persons, carnosine typically adds a mere 7%–10% to their intracellular buffering capacity and neutralizes 2.4–10.1 mmol H⁺·kg⁻¹ dry mass as intramuscular pH declines [35,43]. Yet during exercise-induced metabolic acidosis carnosine is particularly effective as an H⁺ acceptor to due to its imidazole ring and a concomitant shift in its pK_a to 6.83, which is rare for intracellular buffers and vital to maintain pH.

Unlike most buffers, carnosine is potent over a broad pH range in type I and II muscle fibers from the rapid and substantial accrual of intracellular H⁺ and lactate. Carnosine levels range from 4 to 7 and 9 to 13 mM within type I and II fibers respectively [44,45]. Since buffer capacity (β) is a quantitative index of the resistance of a buffer solution to change pH upon added H⁺, its formula is as follows:

β = (Δn)/(ΔpH)     (1)

where Δn represents the incremental additions of proton equivalents to a buffered system and ΔpH is the observed pH changes. This equation is commonly known as the buffer derivative, in which pH (i.e., −log₁₀[H⁺]) is expressed as [H⁺] and where the β in the presence of a neutralizing agent with an acid dissociation constant represented by K_a, is defined as:

    (2)

It is important to note, for near neutral pH’s, the first (K_w/[H⁺]) and second ([H⁺]) terms do not contribute significantly to carnosine’s prowess as a buffer. β can be calculated at pH = 7.1 for a lower limit of 4 × 10⁻³ M carnosine in type I fibers to have a buffer capacity of 9.1 × 10^–4 [42]. At pH = 7.1 for carnosine measured at the higher range found in type II fibers, i.e., at 13 × 10⁻³ M, the buffer capacity rises to 2.95 × 10⁻³, approximately a 3.2-fold increase [42]. At a pH of 6.5, the 4 mM carnosine only slightly changes to 8.7 × 10⁻⁴, and for the 13 mM carnosine to 2.8 × 10⁻³. Thus as pH declines from 7.1 to 6.5 in exercising muscle, carnosine’s buffer capacity is well preserved in type I and II fibers. Not mentioned in the prior reviews [1,2,3,4,5], and unlike buffers and enzymes that only operate within a limited pH range, carnosine’s ability to absorb protons is well maintained over a substantial pH range and makes it a unique and valuable molecule. Thus this paper will now elaborate on the efficacy of prior interventions to raise intracellular carnosine levels.

Consequently research examined the role of oral β-alanine supplementation on exercise performance; which has yielded mixed results. Many β-alanine trials simply examined for the presence of an ergogenic effect [44,46,47,48,49], and could not accurately quantify the full merits of this dietary supplement. One such study randomized college-age men to a placebo or β-alanine treatment with no crossover [49]. β-alanine-dosed subjects ingested an average of 5.2 g·day⁻¹ for four weeks; a subset continued the intervention for ten weeks and consumed an average of 5.9 g·day⁻¹. Before and after each dosing regimen, subjects performed a graded cycle ergometry test to the point of volitional fatigue. While placebo administration did not improve performance, β-alanine-dosed subjects produced significantly more work after four (+7.3%) and ten (+8.6%) weeks of supplementation as compared to baseline values [49]. Yet a far different result was seen from a time trial cycling test, which is a much more sport-specific outcome measure [53]. The time trial study administered concurrent β-alanine (65 mg·kg^–1) and NaHCO₃ (300 mg·kg⁻¹) dosages to elite cyclists; presumably they would act as intra- and extra-cellular buffers respectively, and their combined actions would yield greater ergogenic effects [53]. Yet after a 28-day capsule dosing intervention, results showed placebo-NaHCO₃ and β-alanine-NaHCO₃ time trials improved performance to similar extents [53]. It was implied β-alanine did not improve performance beyond that conferred by NaHCO₃ ingestion [53].

Other studies yielded a myriad of results which appear to be a function of the type of outcome measure examined. The acute effects of a dietary supplement, which included a 4 g dose of β-alanine, were examined for improvements in performance to a series of exercise tests [54]. Twelve recreationally-trained men ingested either the supplement or a placebo. Twenty minutes after ingestion, subjects performed the exercise tests. One week later they received the opposite treatment and performed identical ingestion and test procedures. The supplement produced significant improvements in acute resistive exercise performance at submaxmial loads, perceived energy and physical agility versus placebo administration [54]. It was suggested the supplement delays fatigue produced by strenuous exercise. Yet given the limited evidence that β-alanine offers an ergogenic effect from acute ingestion because intracellular carnosine accrual occurs gradually over time [1,2], performance improvements from the supplement likely resulted from another ingredient in its proprietary blend.

Most other studies examined exercise performance changes from chronic β-alanine intake. β-Alanine (4 g·day⁻¹) or a placebo was given to college wrestlers and football players for eight weeks, with no crossover, to compare changes in exercise performance [55]. Before and after the intervention, subjects engaged in numerous tests related to anaerobic exercise performance. During the intervention both groups continued their customary exercise programs. A comparison of pre- and post-test measurements included non-significant inter-group differences. Yet some test measures showed a trend towards improvement from oral β-alanine intake [55]. It was suggested β-alanine may enhance performance and lean body mass accrual from improvements to the quality of an athlete’s exercise program [55]. However elderly subjects randomized to 12 weeks of β-alanine (3.2 g·day⁻¹) or placebo administration with no crossover noted the former treatment yielded significant pre-post improvements in incremental exercise performance [52]. Yet another study randomized subjects to a 28-day β-alanine (6 g·day⁻¹) or placebo treatment with no crossover to examine responses to an incremental running protocol [56]. Before and after the intervention subjects performed the protocol on a treadmill, which entailed successively more difficult stages until they reached volitional exhaustion. Expressed as a function of onset of blood lactate accumulation (4 mmol·L⁻¹) values, results showed β-alanine supplementation significantly increased heart rate and %VO_2max. In contrast placebo administration elicited non-significant changes for those same variables. However β-alanine-dosed subjects also had a significant decline in their VO_2max expressed in both absolute and relative terms [56].

Additional studies also assessed the presence of an ergogenic effect from oral β-alanine ingestion. With a matched-pairs design and no crossover, men received either an oral placebo or β-alanine treatment [44]. β-Alanine subjects ingested 4 g·day⁻¹ for one week, followed by 6 g·day⁻¹ for the next 28 days. Post-intervention both groups performed a repetitive sprint protocol. As compared to placebo data, β-alanine did not offer an ergogenic effect [44]. A similar lack of improvement to repeated sprint performance occurred at even higher (6.4 g·day^–1) β-alanine doses given over the same time period [57]. It was implied β-alanine did not have an ergogenic effect and that muscle buffer capacity is not truly tested from repetitive sprints of short (15 m) duration [57]. In like fashion, with β-alanine administered at a rate of 4–6 g·day⁻¹ for five weeks as part of double-blind placebo-controlled design, supramaximal sprints (115 and 140% VO_2max) performed to exhaustion did not receive an ergogenic effect from supplementation [58]. Blood lactate levels, collected before and after the sprints, showed non-significant inter-group differences [58]. With similar dosing protocols and study design, β-alanine also did not increase VO₂ max [44,56,59]. However, another trial examined 28 days of oral β-alanine (6.4 g·day⁻¹ for the first six days, then 3.2 g·day⁻¹ for 22 days) on cycling performance with decidedly different results [60]. Before and after the intervention subjects performed an incremental cycle ergometry protocol to volitional failure. Versus placebo data, β-alanine improved performance [60]. With a similar exercise protocol, like results were seen with even smaller (1.6 g·day⁻¹) dosages given over the same time period [49]. The latter studies concluded β-alanine delays H⁺-induced neuromuscular fatigue [49,60].

Another physical activity mode characterized by neuromuscular fatigue from H⁺ accrual is resistive exercise. To examine the presence of an ergogenic effect, after performance of a knee extensor resistive exercise protocol, subjects were divided into a placebo or β-alanine (1.2 g·day⁻¹ for 30 days) group with no crossover [61]. Both groups refrained from physical activity for 30 days, and thereafter repeated the exercise protocol. As compared to pre-treatment values, the interventions yielded no inter-group differences [61]. Yet another resistive exercise study, which used a similar intervention period with a higher (4.8 g·day⁻¹) dosage, noted a different outcome [30]. With a crossover design, strength-trained men underwent separate 30-day β-alanine and placebo treatments [30]. After each 30-day period, subjects performed a six-set 12-repetition squat workout with 90-s rest periods against 70% of their 1 RM load. Versus the placebo condition, β-alanine significantly raised mean power values and was thus deemed to have an ergogenic effect on resistive exercise performance [30].

Since β-alanine generally had an ergogenic impact on anaerobic [30,49,60], but not aerobic [44,59], performance, research also assessed protocols that entailed major contributions from both oxidative and glycolytic metabolism, as some forms of exercise make substantial demands on both energy systems. β-Alanine (2–4 g·day⁻¹) and placebo treatments were compared as part of a double-blind design with no crossover in cyclists [62]. Before and after the eight-week trial, subjects performed a simulated 100-min race on a cycle ergometer [62]. Results showed, as the race concluded with a 30-s sprint, β-alanine significantly raised peak and mean power by 11.4% and 5.0% respectively versus pre-treatment values. In a double-blind study with no crossover, rowers received a placebo or β-alanine (5 g·day⁻¹) capsule dosing treatment [12]. Before and after the 49-day intervention they performed a 2000 m rowing ergometer test [12]. Pre-post changes showed the β-alanine group covered the distance 2.7 s faster, while placebo-dosed subjects were 1.8 s slower. Results also showed a significant correlation between intramuscular carnosine and rowing performance [12].

Other physical activity paradigms were also examined. They include repetitive bouts of exercise done in succession, with little or no rest between successive stages whereby performance is compromised by neuromuscular fatigue. A four-week double-blind study design with no crossover compared β-alanine (2.4–4.8 g·day⁻¹) and placebo treatments given to 400 m sprinters [20]. Subjects performed a five-set 30-repetition isokinetic knee extensor test before and after the intervention. It was shown β-alanine improved knee extensor performance over the last two sets and thus may abate fatigue [20]. β-Alanine and placebo treatments were also compared to note their impact on incremental cycle ergometry exercise [6,63]. When β-alanine was ingested for both 28 (3.2–6.4 g·day⁻¹) and 90 (2.4 g·day⁻¹) days it yielded significant performance gains [6,63]. β-Alanine produced significant gains in ventilatory threshold, physical work capacity to fatigue threshold and time to exhaustion [63]. In contrast placebo-dosed subjects incurred minor changes to the same measures. For the 90-day intervention, pre-post changes entailed ~29% performance gain from β-alanine while the placebo led to negligible changes [6].

β-Alanine supplementation may elevate muscle carnosine [25]; yet its impact as an ergogenic aid is varied. β-Alanine has not improved muscle strength or VO_2max [30,44,51,56,59], yet it raised anaerobic thresholds and delayed fatigue with incremental exercise [6,63]. Carnosine raises exercise performance via greater muscle buffer capacity [8,12], heightened Ca²⁺ release [64], improved troponin C sensitivity for Ca²⁺ [65], reduced reactive O₂ species accrual [66], and vasodilation [67]. There may be several reasons why some studies [9,30,50,60], but not others [8,44,61,68], saw an ergogenic effect. Many trials, due to the mode of physical activity and the type of outcome measured, offered limited insights on β-alanine’s efficacy as an ergogenic aid [1,8,30,44,53,57,59,68]. β-Alanine appears to be efficacious for brief supramaximal exercise immediately preceded by a fatiguing bout of physical activity [62]. Such paradigms evoke sharp intracellular pH losses and thus may benefit from β-alanine supplementation.

Questions about the efficacy of oral β-alanine as an ergogenic aid will persist until the merits of this dietary supplement are objectively and accurately quantified. Until it is, misconceptions about this dietary supplement and its usage will continue. While plausible to help address this issue, muscle buffer capacity measurements are somewhat limited by titration techniques and the feasibility of data collected from exercise [11,33,56]. To avoid misconceptions, new β-alanine research should now focus on simpler measurements, obtained before and after exercise, in order to best assess its ergogenic properties. Since carnosine is most active as a buffer when intracellular H⁺ accrual rates are highest [1,4], exercise that entails multiple supramaximal bouts of activity separated by short rest periods afford the best opportunity to examine β-alanine’s ability to buffer the added proton load. Normally double-blind placebo-controlled within-subject studies, whereby each participant receives multiple treatments and/or dosages, would serve as an ideal study design. However since β-alanine trials are complicated by the very stable nature of intracellular carnosine levels that in turn demand very long (>15 weeks) washout periods to limit carryover effects [12,19], matched-pairs designs, whereby subjects receive only one treatment, are preferable to within-subject studies.

Since lactate increases coincide with higher H⁺ concentrations [40,56], measurements of blood lactate levels over time likely afford the best opportunity to objectively and accurately quantify the merits of β-alanine ingestion, and are superior to prior studies that examined if supplementation merely conferred an ergogenic effect. Blood lactate kinetic investigations have historically entailed a pre- and multiple post-exercise measurements; however such studies included invasive procedures that were uncomfortable to subjects which likely compromised their exercise performance [69,70,71]. In contrast to the invasive measurements [69,70,71], the far simpler and currently popular blood lactate collection from finger tips may do as good a job quantifying the metabolite’s accrual into, and clearance from, the vasculature over time to offer an indirect assessment of buffer capacity. Prior blood lactate kinetic studies entailed supramaximal exercise, the very type of physical activity that evokes high lactate and H⁺ increases [69,70,71]. In theory β-alanine supplementation permits higher work capacities and yield greater peak blood lactate concentrations, due to increased proton buffering. This theory concurs [4,72,73,74] and is in contrast to [1] text from prior reviews that examined the workload-lactate relationship. As assessed through blood lactate kinetics, higher intracellular lactate accrual should also result in a faster rate of entry into the vasculature. It may be hypothesized that β-alanine supplementation will improve intracellular H⁺ buffer capacity and permit greater exercise performance as higher amounts of lactate accrue within, and are removed from, the engaged musculature.