Betaine, also known as trimethylglycine (TMG), glycine-betaine, or oxyneurine, is a neutral zwitterionic compound and a methyl derivative of glycine [1]. Initially isolated from sugar beets in the 19th century, betaine was later on identified in various microorganisms, marine invertebrates, plants, and animals1. Betaine can be acquired from exogenous sources, such as seafood, wheat, and spinach, or synthesized endogenously in the liver or kidney by choline oxidation [2].

Betaine plays several key physiological roles in mammals. First, it acts as an organic osmolyte, protecting cells from osmotic stress such as dehydration and adverse temperatures [2]. It preserves cellular hydration while maintaining integrity of macromolecular functions and regulates fluid balance and trans-epithelial water movement [3], thereby assisting in cell volume regulation. Second, betaine protects proteins from urea-induced denaturation by preventing the formation of folding intermediates that promote the loss of enzymatic structure and function [4]. Moreover, as an osmolyte, betaine has been demonstrated to enhance water structure and promote tighter protein folding, which may further contribute to its stabilizing effects on protein conformation [5]. Third, betaine acts as a methyl donor in the methionine cycle by donating a methyl group to homocysteine via betaine-homocysteine methyltransferase, leading to the formation of methionine and supporting the synthesis of S-adenosylmethionine, a central methyl donor in cellular metabolism [6]. The increased availability of S-adenosylmethionine, in turn, stimulates the synthesis of key biological compounds, such as creatine [7] and carnitine [8]. These physiological properties have led to the increasing recognition of the clinical uses of betaine, including supporting the normal function of vital organs and mitigating the development of various diseases, such as hyperhomocysteinemia [1,2].

In addition to its therapeutic effects, studies have explored the ergogenic potential of betaine on exercise performance, in light of these physiological mechanisms. The International Society of Sports and Nutrition (ISSN) defines ergogenic aids as mechanical, nutritional, pharmacological, or psychological techniques or tools that enhance the exercise capacity or training adaptation [9]. Particularly, dietary supplements are deemed ergogenic if most human studies have demonstrated their effectiveness in promoting muscle hypertrophy or improving performance through exercise training [9].

One such performance outcome is endurance, which has been referred to in one study as “endurance intense efforts [10],” and is defined as the ability of the cardiorespiratory system to supply energy for exercise bouts lasting longer than 1 minute, with a preponderance of the “oxidative phosphorylation pathway” [10]. Betaine has been proposed to enhance endurance through its electrophilic methyl group (EMG) moiety [11]. This mechanism involves the acceptance of a nucleophilic hydride ion (H-) from NADH, resulting in the oxidation of NADH to NAD+ and the formation of methane (CH4) [12]. NAD+ is an intracellular coenzyme that plays a vital role in driving ATP regeneration via glycolysis and the citric acid cycle [13]. Supporting this mechanism, a previous cell culture experiment demonstrated that betaine treatment increases mitochondrial potential, leading to higher ATP levels [14]. Furthermore, betaine has been suggested to upregulate the activity of cytochrome c oxidase, a terminal enzyme in the mitochondrial electron transport chain, thereby promoting mitochondrial functioning [15]. Similarly, one study proposed that betaine may facilitate lactate oxidation by enhancing mitochondrial respiration [16], potentially contributing to increased ATP production. Several studies have reported a lower [16] or no increase [17] in plasma lactate concentrations after exercise, while demonstrating a significantly greater work output following betaine supplementation. Furthermore, betaine supplementation has been suggested to protect citrate synthase, a rate-limiting enzyme in the citric acid cycle, from thermal and urea denaturation [18].

Given these proposed mechanisms, betaine may be a promising ergogenic aid for athletes and individuals engaged in endurance activities. However, only clinical trial evidence supports the true performance benefit of betaine on endurance performance. Previous systematic reviews have evaluated the ergogenic effects of betaine supplementation on muscle strength, power [19], and body composition [20]. A recently published systematic review [21] examined the effects of chronic betaine supplementation across a broad spectrum of exercise performance outcomes, including aerobic capacity. However, their analysis was largely confined to conventional physiological markers such as oxygen uptake (VO₂), maximal oxygen consumption (VO₂max), and heart rate. Although these metrics are commonly used to characterize aerobic performance, they do not fully capture the complexity of endurance performance in a real-world setting. Some studies indicate that aerobic capacity alone does not dictate endurance success, as prolonged or high-intensity efforts often demand substantial anaerobic contributions [22,23]. Moreover, real-world competitions involving surges, sprint finishes, or overtaking maneuvers underscores the pivotal role of anaerobic performance, which is not accounted for by traditional aerobic metrics. In contrast, the present systematic review addresses this gap by providing a more targeted and integrative evaluation of endurance outcomes, incorporating both physiological measures and performance-based indicators, such as time-to-exhaustion (TTE), time-to-completion (TTC), and power output, thereby comprehensively assessing how betaine supplementation may influence endurance performance in practical and competitive contexts.

Therefore, this systematic review aims to evaluate the ergogenic effects of betaine supplementation on endurance exercise performance in healthy individuals, using a combination of physiological measures and performance-based outcomes. Health professionals, coaches, endurance athletes, and active individuals engaging in endurance activities, and other stakeholders would gain insight into an alternative performance enhancer that is considered safe [24], rapidly absorbed [25], well-tolerated, and cost-effective [26].

This systematic review evaluated the effectiveness of betaine supplementation on endurance exercise performance. Five studies (three parallel-design and two cross- over-design) involving 101 participants were included, with intervention periods ranging from 14 to 98 days and betaine doses between 1.25 to 5 g/day. Although only two studies [31,32] demonstrated significant improvements in endurance performance (VO₂max and mean power), research on betaine remains limited, with all included studies exhibiting a high risk of bias. This is consistent with previous reviews that have explored the ergogenic potential of betaine in aerobic endurance exercises, by examining its effects under conditions of chronic supplementation duration [11] or high-temperature environments [21]. The findings of these studies underscore the paucity of high-quality research which challenges the ability to draw definitive conclusions regarding the effects of betaine on endurance exercise performance.

Among the four studies [29,31-33] evaluating physiological measures, only one [31] demonstrated significant improvements in VO₂max following betaine supplementation. This finding may be attributed to the longer supplementation period (98 days) in that study, compared to the shorter, acute regimens of 4 to 14 days in the other three studies [29,32,33]. A previous trial reported significant improvements in bench press work capacity following 6 weeks of betaine intake [34], suggesting that sufficient intramuscular betaine accumulation may require a longer duration to potentially elicit performance benefits. This is further supported by findings from another trial that observed elevated plasma betaine concentrations after 4 weeks of daily supplementation [35], reinforcing that a prolonged intake period may be necessary to achieve adequate tissue saturation and produce a physiological impact. Therefore, it may be advisable to prescribe betaine supplementation for a period of 4 to 6 weeks to enhance endurance performance, although additional high-quality research with well-controlled, rigorous designs, and adequate sample sizes is required to substantiate this recommendation.

Similarly, among the four studies [29,30,32,33] assessing performance tests, only one32 reported a significant increase in mean power during the final stage of the Wingate Anaerobic Test following betaine supplementation. During this stage, the fatigue index was notably lower than that in the pre-supplementation phase, and the ratings of perceived exertion remained unchanged, indicating enhanced performance without an increase in perceived effort. The mechanisms underlying these findings may be linked to the effects of betaine on the central nervous system. Betaine has been hypothesized to mitigate fatigue by increasing the availability of free choline via its proposed role as a methyl donor in phosphatidylcholine synthesis, thereby potentially supporting acetylcholine production in motor neurons [11]. Enhanced acetylcholine synthesis may sustain the central drive, facilitate continuous motor recruitment, and thereby maintain power output. Additionally, teleoanticipation, arising from the closed-loop nature of the Wingate Anaerobic Test, enabled participants to strategically regulate energy expenditure [36], which may have contributed to the increased power output observed in the final sprint in Waldman et al (2023). Consequently, closed-loop endurance sports such as track running, track cycling, swimming, and speed skating, as well as long endurance sports that involve intermittent bursts of explosive movements, such as soccer, may benefit from this supplement. However, further studies are needed to confirm these findings.

All included studies were assessed as having a high risk of bias, primarily due to deviations from the intended interventions. None of the studies reported adequate blinding of participants or outcome assessors, raising the possibility that the observed effects may have been influenced by deviations from the protocol rather than by the intervention itself. Similar concerns have been highlighted in a previous systematic review on betaine supplementation [37]. Such methodological limitations and reporting deficiencies are prevalent in nutrition research [38,39] and contribute to reduced confidence in the validity of findings from betaine trials.

Further concerns were identified in the randomization process. Specifically, none of the studies described how the allocation sequence was generated or concealed, and only two reported baseline characteristics, thereby limiting the ability to ensure group comparability and increasing the risk of confounding. Selective reporting bias also raised concerns, as none of the studies provided pre-specified protocols or statistical analysis plans. This lack of transparency raises the possibility that the outcomes were selectively reported, potentially overstating the benefits of the intervention.

In contrast, the risk of bias was considered low in the domains of missing outcome data and measurement of outcomes. Although no statistical corrections were reported, the extent of missing data appeared minimal and was unlikely to introduce systematic bias. The outcome measures employed were appropriate and consistently applied across groups. Although the blinding of the outcome assessors was not reported, the objective nature of the outcomes suggests that measurement bias was unlikely.

Taken together, these methodological limitations warrant a cautious interpretation of the pooled results and highlight the need for rigorously designed and transparently reported trials to more accurately assess the ergogenic effects of betaine supplementation. To address these concerns, it is essential to promote adherence to established reporting guidelines, such as the CONSORT statement, along with the forthcoming nutrition-specific extension [39]. Furthermore, research institutions, publishers, and funding bodies should actively support these efforts by incentivizing compliance, strengthening reporting infrastructure, and providing targeted training to improve the quality and transparency of trial reporting [40].

This systematic review had certain limitations. First, meta-analyses and meta-regressions were not conducted due to the inclusion of fewer than 10 studies examining endurance outcomes, underscoring the need for additional studies with larger sample sizes to enhance the robustness and generalizability of findings. Second, the inconsistent findings and a lack of key themes in variables such as population, age, training ability, and supplement dosages highlight the need for further investigation to elucidate patterns and develop targeted recommendations for specific populations and contexts. Notably, the participant age heterogeneity was high (82.11%); however, the limited number of studies hindered the identification of clear trends, emphasizing the necessity for additional studies that would enable subgroup analyses or meta-regressions to better account for variability. Third, substantial methodological variability in the assessment of endurance capacity was observed across the included studies, highlighting the need for future research to adopt more practical and physiologically relevant approaches. Measures such as VO₂max, lactate threshold, and movement economy are increasingly recognized as key determinants of endurance capacity [22,41], and their inclusion along with widely accepted protocols, such as laboratory-based graded exercise tests [42], may enhance comparability across studies. Field-based assessments are also commonly utilized in sports settings because of their practicality, accessibility, and adaptability to sports-specific demands. Common examples include the multi-stage 20-m shuttle run test [43,44], particularly among adolescents and young adults, as well as the step test [45,46] and Cooper’s 12-minute run test [47]. However, these tests are generally considered to be less precise than laboratory-based assessments and should be interpreted with caution when evaluating endurance outcomes. Finally, pre-trials should be conducted to thoroughly investigate the mechanisms through which betaine supplementation influences endurance performance, thereby offering deeper insights into its potential benefits.

In conclusion, this systematic review highlights the limited and inconclusive evidence regarding the definitive ergogenic effect of betaine supplementation on endurance exercise performance. Only two of the five included studies reported significant improvements in VO₂max and mean power, suggesting that any potential benefits of betaine may be context-dependent, particularly under conditions involving prolonged supplementation or closed-loop endurance activities. The interpretation of these findings is further limited by the substantial variability in the population characteristics, dosing protocols, assessment methods, and the consistently high risk of bias across all studies. Nonetheless, the exploratory findings related to central drive and fatigue perception present a promising avenue for future research, suggesting that betaine may confer benefits in endurance sports involving by intermittent bursts of explosive movements. However, these potential effects require further investigation. Future research should incorporate larger sample sizes, consistent endurance performance assessments, extended supplementation durations, and adherence to tools that ensure methodological rigor in clinical trials, in order to definitively ascertain the ergogenic potential of betaine and provide tailored recommendations for specific populations and performance contexts.