Effects of a 29-Day Beta-Alanine Supplementation on Contractility and Recovery of the Musculus Rectus Femoris

Beta-alanine supplementation has been shown to improve fatigue-associated alterations in Ca2+ sensitivity and handling, thereby possibly delaying fatigue and promoting recovery after exercise. Some in vitro data further indicate that beta-alanine positively affects muscle contractility.
In this double-blind study, 24 physically active participants were randomly assigned to a placebo (n=12) and an intervention group (n=12). They performed an isokinetic exercise protocol consisting of 15 concentric and eccentric all-out contractions per set at ω=60°/s, ROM=0-170°, hip angle=110° and with a 2 min interset rest period until concentric force dropped below 50% of the predetermined peak force. Muscle contractility was tested during the interset rest periods and during a 24 h recovery period using Tensiomyography.
No significant interaction effects for time group were found, indicating that beta-alanine did not improve work output, fatigue, or muscle contractility during or after maximum effort isokinetic knee extension.
The lack of findings could be explained by exercise duration, type of exercise, dosing protocol, training status or a priori power analysis.

Key Words: Isokinetic Exercise, Muscle Contractility, Muscle Recovery, Tensiomyography

In recent years, the amino acid beta-alanine has repeatedly drawn the attention of exercise physiologists and athletes from various disciplines. Today it is one of the most popular sport supplements. The foundation of this interest is, among other things, rooted in the fact that prolonged oral supplementation of beta-alanine can significantly increase concentrations of the dipeptide carnosine, which is abundant in skeletal muscle (21). So far, literature reviews have linked these increased intramuscular concentrations of carnosine to improved athletic performance (6, 34).
Beta-Alanine is a non-essential amino acid that is, in small amounts, endogenously synthesized in the liver but enters the body mainly through diet, especially through the consumption of meat, poultry, and fish (21, 37). Carnosine can be formed in the body from beta-alanine and L-histidine, which is found in particularly high concentrations in muscle cells (21). The synthesis of carnosine occurs in the muscle in situ by carnosine synthase: Adenosine triphosphate + L-histidine + beta-alanine → Adenosine monophosphate + diphosphate + carnosine (20). The rate-limiting factor in carnosine synthesis is the availability of beta-alanine (3). To effectively raise carnosine concentrations and potentially increase athletic performance, supplementation of 3.2-6.4 g beta-alanine per day over a 2–4-week period is recommended (34), where large increases in carnosine concentrations (+91.1%±29.1%) have been observed in non-vegetarian and physically active men (42). Among omnivores differences in muscle carnosine content with high vs. low beta-alanine intake could not be identified (14).
The systematic review and meta-analysis by Saunders et al. (34) identified exercise duration as the greatest influencing factor in the effectiveness of beta-alanine supplementation, with the authors supporting its efficacy for efforts lasting between 30 seconds and 10 minutes. However, another meta-analysis found no significant effects of beta-alanine for exercise durations shorter than 60 seconds (22). Performance-enhancing effects of elevated carnosine levels through beta-alanine supplementation are attributed to several factors that delay fatigue and postpone exercise termination.
Although the influence of hydrogen (H+) ions on fatigue is still debated (30), carnosine is known to act as a buffer for these ions, and thus delays acidosis. Even if H+ ions may not be the primary explanatory factor for muscular fatigue, it is plausible that beta-alanine can reduce fatigue via this pathway. Furthermore, antioxidative properties of carnosine reduce the negative effects of reactive oxygen species (4, 13) on the calcium (Ca2+) sensitivity of the contractile apparatus and Ca2+ reuptake of the sarcoplasmic reticulum (13). Additionally, carnosine seems to have attenuating effects against accumulations of inorganic phosphates (Pi), which occur during repetitive contractions and can impair sarcoplasmic reticulum (SR) release, as well as Ca2+ sensitivity of myofibrillar proteins (2). There is also evidence of a beta-alanine induced shortening of the relaxation time by improving Ca2+ reuptake of the SR (17, 24).
In summary, beta-alanine shows numerous positive effects on muscle function. However, data on the effects of beta-alanine supplementation on muscle contractility are still lacking. The purpose of this study was therefore to investigate the effects of a 29-day beta-alanine supplementation on the contractile properties of the rectus femoris muscle, throughout and after isokinetic exercise.

Participants
An a priori power analysis using GPower 3.1 (University of Düsseldorf, Germany) revealed that 24 subjects were needed to detect a medium effect (f=0.3) with an 80% chance (power=0.8) and an α=0.05. Therefore, 24 participants were recruited to participate in the study (beta-alanine: n=12, 6 female, 6 male, aged 21-30 years, body mass=71.83±11.01 kg, height=1.75±0.10 m, BMI=23.41±2.51 kg/m2; placebo: n=12, 6 female, 6 male, aged 22-30 years, body mass=68.69±9.97 kg, height=1.72±0.09 m, BMI=22.97±1.52 kg/m2). Figure 1 illustrates the flow of participants. The subjects consisted of recreational athletes (n=7), sports students (n=10), and competitive athletes (n=7) who engaged in a wide variety of disciplines (team athletes, strength athletes, divers, sailors, swimmers, cyclists). Participants were enrolled in the study, if they had not taken any performance-enhancing supplements within the six months prior to study entry, if they were free of medical restrictions with the potential to interfere with the performance within the study, they did not follow a vegetarian or vegan diet, had no intolerances to fructose, sorbitol, or beta-alanine, were not diagnosed as diabetic, were not taking any medications, and were physically active for at least 150 minutes per week. Over the study period, all participants were asked to maintain their dietary and training habits. They expressed their agreement by signing a consent form. This study involving human participants was reviewed and approved by the local ethics committee (#2021-59, Goethe University Frankfurt, GER) and was conducted in accordance with the ethical standards set by the declaration of Helsinki.

Randomization 
24 participants were randomized (block randomization) into an experimental and a control group by a person not involved in the experiment using statistical software (BiAS, version 11.12, epsilon-publishing). The result of this randomization were two groups with 12 subjects in the placebo group and 12 participants in the experimental group. Both groups consisted of n=6 males and n=6 females.

Double Blinding
To ensure double blinding, a person not involved in the experiment packed the supplements in envelopes, which the investigator handed out to the subjects on the first day of the pre-test. 

Design of the Experiment
To explore the effects of 29 days of beta-alanine supplementation on the contractile properties of the rectus femoris muscle during and after an isokinetic exercise protocol, a double-blind, placebo-controlled, repeated-measures group design was conducted. The experimental procedure is illustrated in figure 2.

Study Design

Familiarization
All measurements were conducted at Goethe University in the laboratory of the Department of Exercise Physiology and Sports Medicine.  Participants first completed a familiarization session on the isokinetic dynamometer used in the experiment (IsoMed 2000; D&R Ferstl, Hemnau, Germany) to get accustomed to the machine and the isokinetic exercise protocol and to minimize bias due to potential learning effects (29, 33). Participants first warmed-up (described in more detail below) on a bicycle ergometer. Following the general warm-up, participants were asked to sit on the isokinetic dynamometer as depicted in figure 3. The rotational axis of the dynamometer was aligned orthogonally to the dominant leg at the lateral condyle. The dominant leg was determined using the method of Van Melick et al. (40) with the question “If you were shooting a ball at a target, which leg would you use to shoot the ball?”. The isokinetic dynamometer was set as shown in figure 3. Hip angle: 110° (180° supine position), range of motion (ROM) of the knee joint: 90-170° (180° full extension), mode: eccentric-concentric, acceleration: very fast, deceleration: hard. According to recommendations of the review by Undheim et al. (38), the angular velocity was ω=60°/s, as the authors summarized that the torque output decreased at higher angular velocities and the maximum torque output was reached between 0 and 60°. The lower edge of the lever arm dual pad was placed two finger widths above the lateral malleolus. The thigh and hip were secured with straps and the shoulders were fixed with adjustable pads to prevent body position slippage (23). Gravity compensation of the dominant leg was performed after the lever arm of the dynamometer was in horizontal position, with the participant fully relaxing the leg. During the exercise, the participants held on to handles which they were allowed to adjust so that they could hold on to them comfortably. All settings made were saved and replicated during subsequent measurements. Since the angle of the isokinetic lever arm did not exactly correspond to the actual angle of the knee joint, the knee angle of the supine Tensiomyography (TMG) measurements (120°) was transferred to the angle of the isokinetic lever arm using a goniometer. This ensured a knee angle of 120° during the interset breaks, where seated TMG measurements were performed. The center of each goniometer end was aligned with trochanter major and lateral malleolus. The angular position of the knee was noted from the dynamometer display and saved for later measurements. A minimum of 48 hours elapsed between familiarization and pretest.

Pre- and Post-Test Procedure
During the pre-test, the contractility of the rectus femoris muscle was recorded with TMG on a treatment bench in supine position (TMG supine). Afterwards, participants generally warmed-up on a bicycle ergometer and were seated on the isokinetic dynamometer. There, the TMG parameters were again recorded in TMG seated and the knee was then prepared for exercise in a specific warm-up. The contractile properties were recorded again after the specific warm-up (TMG seated) and after the maximum voluntary torque (peak torque) of the subjects was determined. Thereafter, the fatigue protocol was performed on the isokinetic dynamometer and TMG measurements were taken during set breaks (TMG seated) to monitor immediate signs of fatigue. After completion of the fatigue protocol, participants were asked to lie down on a treatment bench. There, in a supine position, another TMG measurement was taken immediately after exercise (TMG supine). To evaluate the contractile behavior of the rectus femoris muscle during the recovery phase, additional TMG measurements were recorded at one hour post exercise and 24 hours after the first TMG measurement (TMG supine).
At the end of the 29-day supplementation with beta-alanine or a placebo, the procedure just described was repeated in the post-test. Participants were instructed to refrain from strenuous exercise, caffeine, and alcohol for 24 h prior to testing.

Exercise Protocol

Warmup
A general warm-up was performed on a bicycle ergometer (marked 
as GWU in figure 2), on which participants pedaled with two watts per kilogram of body weight, at a frequency between 60 and 70 rounds per minute, for five minutes duration. Following the general warm-up, participants were asked to sit on the isokinetic dynamometer. Applying the settings described in the familiarization section, the knee joint was once again specifically warmed up (marked as SWU in figure 2) with 2x10 repetitions of successively increasing (very light - maximum effort in the 10th repetition) voluntary muscle contraction. The pause time between sets was set to two minutes.

Determination of Peak Torque (PT)
After local warm-up, peak torque was determined on the isokinetic dynamometer. For this purpose, five repetitions of maximal voluntary contraction in the eccentric and concentric phases were recorded (19). During testing, subjects were encouraged to ensure the best possible performance (15, 28). This was done in the form of loud shouting and clapping. The largest recorded concentric value was taken as the peak voluntary torque.

Fatigue Protocol
After the warm-up and the determination of the peak voluntary torque, the fatigue protocol was performed on the isokinetic dynamometer. One set consisted of 15 eccentric and concentric repetitions of maximal voluntary contraction. Participants received encouragement (clapping and shouting) to ensure their maximal effort (15, 28). After a set was completed, the thigh belt was unfastened during the two-minute set break, the programmed protocol was interrupted to allow the leg to be adjusted to an angle of 120 degrees, and two TMG measurements with of the rectus femoris muscle were performed with an electrical stimulus of 100 mA and an interval of 30 seconds between electrical stimuli. The belt was then reattached and after the rest of 2 minutes had elapsed, the next set of the loading protocol was started. This procedure was repeated until the termination criterion was reached. The fatiguing protocol was terminated when the subjects were no longer able to apply more than 50% of their peak torque in three consecutive repetitions. This allowed the same fatigue to be achieved for each subject individually. Participants remained seated on the isokinetic dynamometer throughout the exercise protocol, including rest periods, and were required to hold the dynamometer handles while doing so.

Tensiomyography (TMG)
Figure 4 shows the TMG parameters analyzed. In our experiment, TMG measurements were performed in two different body positions. The classical TMG measurement in the rectus femoris muscle is conducted in supine body position (TMG-supine). We adapted this method to make our results comparable to research following the same standards. The switch to the seated body position (TMG seated) was needed to investigate acute rectus femoris contractility changes, brought about by isokinetic exercise. Time was not sufficient to conduct these measurements in supine position, which is why we decided to measure directly on the isokinetic dynamometer, in seated position. The procedure in each type of recording will be described in more detail below.

TMG Measurements in Supine Position (TMG-supine)
Measurements in supine position were taken on the dominant leg in pre-test and post-test before the start of the general warm-up, immediately after the termination of the exercise protocol, one hour after the termination of the exercise protocol and 24 hours after the first TMG measurement. This was done according to Rey et al. (31) at 120 degrees of knee flexion (180 degrees corresponds to full extension of the knee) supported by a foam pad. The sites where the sensor and electrodes were placed were shaved and cleaned with alcohol. The TMG sensor was placed perpendicular to the muscle belly to record the radial displacement (Dm) of the rectus femoris muscle. The position of the sensor and electrodes was located according to Roth et al. (32) and marked with a pen. To find the same location during subsequent measurements, subjects were instructed to continuously redraw the location during the intervention period if it faded, for example, due to contact with water. The 5x5 cm electrodes (self-adhesive Dura-Stick Plus, 50x50 mm) were placed symmetrically to the marked sensor point with an inter-electrode distance of 5 cm (32), with the red cable attached proximally and the black cable distally. The sensor was then placed on the marked point perpendicular to the muscle belly and pushed back approximately 2 cm into the shaft (32). If necessary, the touchdown point of the sensor was still slightly changed to place it on the point with the largest muscle belly (35). The spring constant of the sensor was 0.17 N/mm (27). The stimulus pulse lasted for one millisecond and the stimulus was increased from 20 to 100 mA in 10 mA steps (25, 41) until a maximum response was visible (figure 5). Rests of 30 s were inserted between successive stimuli to minimize the effects of fatigue and potentiation (35).

TMG-Seated Measurements
The TMG measurements before the isokinetic warm-up (SWU), after isokinetic warm-up, after peak torque evaluation, and during set breaks of the isokinetic fatigue protocol (S1-Sn), while participants were seated on an isokinetic dynamometer. During the breaks, the thigh strap was released, the knee angle was brought to 120° (180° full extension), the sensor was pointed at the marked point, a contraction was elicited by a stimulus of 100 mA, the sensor angle was then changed slightly, and after an interval of 30s, a second stimulus of 100mA was elicited and another curve recorded. The thigh strap of the isokinetic dynamometer was then reattached, and the knee was moved to the starting position of 170° in preparation for the next set. At the end of the two-minute rest, the next set of the loading protocol began. An overview of the measurement time points is displayed in figure 2, where TMG-seated is marked as TB.

Supplementation
All participants supplemented 3.2 grams of beta-alanine or a placebo (mannitol), depending on the group allocation. The dose was set at 3.2g of beta-alanine per day to achieve a sufficient increase in muscle carnosine (21, 34, 42) but to avoid paresthesia (10), which is the only known side effect (12) that could have compromised the double-blinded design. The risk of paresthesia was further reduced by taking beta-alanine and placebo in four equal doses (0.8 g) - administered in the form of capsules, at least 3-4 hours apart (34). The investigators recommended ingestion at breakfast, lunch, and dinner, as ingestion of beta-alanine in combination with a meal was found to be effective in increasing muscle carnosine levels (37). Furthermore, it was recommended that the last dose be ingested before sleep. Participants were asked to photographically document their intake and send the pictures to the investigators so that intake patterns could be tracked. Since it proved difficult for some participants to record their intake photographically in their daily work routine, written documentation was also accepted. Capsules were prepared by an investigator using a capsule filling machine to be able to administer the desired dose. To allow for the double-blind design of the study, capsules were packaged in envelopes, according to randomization, by a person not involved in the experiment and handed to the investigator before the start of the measurement session. Participants were excluded from the study if they forgot to ingest more than 4 capsules (3.45% of the total dose).

Statistics and Computations 
The statistical analysis was performed with the general-purpose programming language Python (version 3.11.3). The detailed analysis can be followed in a Jupyter Notebook, designed to share live code and visualizations, which is provided as supplementary material to this study.

Isokinetic Data
The isokinetic data were extracted from the Isomed hard drive and saved as CSV files. The raw Isomed data for each subject were merged into a single CSV file. For each subject, the work performed for each phase of the isokinetic protocol was calculated as torque integral over the complete range of motion, and fatigue indices were computed as (mean of peak three torque values - mean of last three torque values / mean of mean of peak three torque values * 100). For each unique participant differences in work done and fatigue index were calculated by subtracting pretest values from posttest values. These unique differences were then statistically compared between groups by a t-test.

TMG Data
For every measurement time point in supine and seated TMG measurements only the time-displacement curve with the largest displacement was kept for analysis (27). These raw data were combined into a single pandas dataframe and TMG parameters were calculated from scratch by an algorithm according to definition to increase their accuracy, as especially parameters Ts and Tr could not always be precisely determined by the precalculated values of the manufacturer’s result sheet. In every case, the first displacement peak value was defined as Dm. Contractile velocity Vc was calculated as (Dm/(Td+Tc)) (26). In TMG supine individual contractility parameter changes were calculated as difference between the same time point of pretest and posttest (e.g. supine 1 measurement of posttest subtracted by supine 1 measurement of pretest). In -TMG-seated, since the subjects completed different numbers of sets (S1-Sn) of the exercise protocol until the termination criterion was reached individual differences of each participant were calculated between the values obtained after completion of isokinetic warmup and after termination of the exercise protocol. In both cases individual contractility parameter changes between pretest and posttest were statistically compared by mixed ANOVA analysis.

Data Analysis
Data of 24 (n=beta-alanine, n=12 placebo) participants were analyzed according to their preassigned group allocation. The data from excel/csv files were parsed and transferred to a pandas data frame. After cleaning the data, by adjusting naming conventions and datatypes, descriptive statistics followed, inspecting the sample for age, weight, height, BMI and sex distribution by group and plotting the results. 
For each TMG parameter in its specific measurement condition (seated and supine) a mixed ANOVA was calculated using the open-source package pingouin (39) to examine possible differences in muscle contractility, with “group” (placebo vs. beta-alanine) as between-subjects factor and “time” (differences in measurement time points) as within-subjects factor. For calculations in pingouin, correction was set to ‘auto’ to compute Mauchly’s test of sphericity to determine whether p-values needed correction. If so, a Bonferroni correction was applied (parameter Tr). Effect size was calculated as partial eta squared.
The data can be accessed through a public GitHub repository. The URL is found in Data availability section at the end of this article.

Pre to Post Differences of TMG Parameters Measured in Supine Position
For the supine position, every mixed ANOVA conducted for differences in the changes of the six TMG Parameter from pretest to posttest did neither show interaction effects between time*group nor group effects nor time effects (table 1, figure 6). pGG values indicate Greenhouse-Geisser sphericity corrected p values.

Pre to Post Differences of TMG-Seated Parameters
For the seated position, every mixed ANOVA conducted for differences in the changes of the six TMG Parameter from pretest to posttest did neither show interaction effects between time*group nor group effects nor time effects (table 2, figure 7).
We therefore conclude that in this study beta-alanine supplementation did not alter muscle contractility when compared with placebo intake. Thus, our hypothesis, that beta alanine might alleviate detrimental effects of fatigue on muscle contractility, is rejected.

Pre to Post differences of Isokinetic Work and Differences of Fatigue Index
An independent one-sided t-test did not reveal differences between pretest to posttest changes in work done between beta-alanine and placebo group (t(22)=-0.321, p=0.376) (figure 8). Changes of fatigue from pretest to posttest, as calculated by differences of fatigue indices, were not significantly different between experimental and intervention groups as an independent one sided t-test displayed t(22)=1.073, p=0.853. We therefore conclude that beta-alanine supplementation does not increase isokinetic work output or reduce fatigue as measured by fatigue index.

The purpose of this study was to investigate if supplementation of beta-alanine had positive effects on the contractile properties of the rectus femoris muscle throughout and after all-out isokinetic knee extensions that may delay fatigue, enhance performance, and improve recovery. For a power revealing medium effects with 80 % probability, the key findings are that no interaction effects for time*group could be discovered in any of the TMG contractility parameters recorded, nor in work output, indicating that beta-alanine did not delay fatigue, enhance performance, or contribute to faster muscle recovery. Our hypothesis is therefore rejected.

Exercise Duration
A possible reason why we did not observe interaction effects may be related to the time span of the fatiguing sets performed in this study, which took about 48 seconds each. The systematic review and meta-analyses by Saunders et al. (34) identified exercise duration as the greatest influencing factor regarding the efficacy of a beta-alanine supplementation. In 2016, the authors suggested that effects were greatest for a time frame between 0.5-10 minutes (34). A former meta-analysis by Hobson et al. (22) found no benefit for beta-alanine supplementation when exercise duration was less than a minute. Other studies involving isokinetic load protocols support the idea that effects may be larger, when exercise duration is longer than the one chosen in this experiment. Measuring trained sprinters, Derave et al. (11) found attenuated fatigue in the beta alanine group in set 4 and 5, using a 5 x 30 repetition MVC exercise protocol. Glenn et al. (16) found improvements in peak torque and work done for female masters’ athletes, using a 50 repetition all out exercise protocol. In comparison to studies with a similar exercise duration, results seem to be controversial and rather uncertain, although overall effects favor beta-alanine supplementation in the time span of exercise chosen in this study (34). That being said, we would like to point out that in the meta-analysis by Saunders et al., confidence intervals for mean effect sizes in 32 out of 41 analyzed cases (0.5 to 10 minutes of exercise duration) cross the line of no effect or do not favor a beta-alanine supplementation. For this reason, we consider it appropriate to be cautious when evaluating the overall effect of a beta-alanine supplementation. In summary, our observations fall within a period where the effect of beta-alanine has not yet been sufficiently confirmed.

Exercise Intensity and Calcium Sensitivity
When interpreting our results, it is crucial to mention that the primary mechanism by which beta-alanine is supposed to help delay fatigue during exercise is still not fully understood since the magnitude of the influence of H+ ions on fatigue is still a matter of debate8. For this reason, the primary mechanism of beta-alanine has been attributed to either its buffering capacity against H+ ions (22, 34) or the improvement of Ca2+ handling/sensitivity (13).
Since beta-alanine both buffers H+ ions (1, 21) and improves Ca2+ handling (13), supplementation should theoretically have a positive effect on muscular fatigue regardless of the fatigue mechanism. Due to the duration and intensity of the exercise in our experiment, it can be assumed that it was associated with metabolic acidosis caused by a strong increase in ATP hydrolysis (18). Since we could not detect any positive influences of beta-alanine, we assume that H+ did not contribute as largely to fatigue in our experiment as Ca2+ sensitivity did. However, when exercise intensities are maximal, a decrease in Ca2+ sensitivity leads to smaller reductions of force output than with moderate intensities (2). The beneficial effects of beta-alanine on Ca2+ sensitivity might therefore be more obvious in submaximal testing conditions when the influence of Ca2+ sensitivity is stronger.

Dosing Protocol
With 3.2 g beta-alanine per day, we supplemented at the lower end of the dose recommendation range. This dosage was chosen to minimize the occurrence of potential paresthesia - which could have compromised the double-blind design, while still effectively increasing carnosine levels as shown previously (6, 16, 34). Although there is evidence that increases in muscle carnosine concentrations are dependent on the total dose ingested and the duration of the supplementation period (5, 36), we have no reason to believe that a higher dosing protocol would have resulted in different observations in work output since evidence from a meta-analysis concludes that neither the total amount ingested nor the supplementation duration mediated the effect of beta-alanine supplementation on exercise outcomes. These conclusions may have been influenced by the wide range of dosing protocols (3.2-6.4 g/day) analyzed (34).
However, we expected to notice beneficial changes in muscle contractility by carnosine mediated increases in Ca2+ sensitivity of the contractile apparatus and a possible improvement in Ca2+ release of the SR in Type 1 fibers (13), that seem to have a strong correlation to the contraction time (Tc) measured in Tensiomyography (7, 8, 35, 43). The reasons for this observation remain unclear.
A major limitation of this study is the absence of direct verification of intramuscular carnosine content (via ¹H-MRS or muscle biopsy). While such methods can confirm physiological efficacy of supplementation, muscle biopsies are painful and may hinder recruitment. ¹H-MRS is expensive and was beyond the financial scope of this project. Instead, we relied on a dosing protocol recommended by a systematic review and meta-analysis to reliably increase muscle carnosine (34).

Training Status
Although there is contrasting evidence (9), another factor influencing the effects of beta-alanine seems to be the training status of the individuals. The systematic review and meta-analysis by Saunders et al. concluded that effect sizes tend to be smaller in trained individuals, although these modest changes may still translate into meaningful performance improvements in competitive settings (34). Our sample consisted of a variety of different disciplines and training levels (recreational to competitive athletes). While this reflects real world beta alanine use across various athletic populations, our heterogeneous sample may have introduced variability in responsiveness and may therefore be another factor for the lack of findings in this study. However, given the randomized group allocation, we assume that potential differences in training status were distributed equally across conditions, which should have balanced these differences between groups, thereby reducing the risk of confounding.

Power-Analysis
Our a-priori power analysis predetermined a sample size of 24 participant based on a medium effect of f=0.3, α=0.05 and a power of 0.8. While the present study did not reveal significant interaction effects, this does not eliminate the possibility that a smaller, yet relevant effect may exist.

In conclusion, our study suggests that beta-alanine does not improve work output, fatigue, or muscle contractility during or after maximum effort isokinetic knee extension.

Conflict of Interest
The authors have no conflict of interest.

Funding
This research did not receive any funding.

Beta-alanine affects fatigue-associated alterations in Ca2+ sensitivity and muscle function. The purpose of this study was therefore to investigate the effects of a beta-alanine supplementation on the contractile properties of the rectus femoris muscle. 24 physically active participants performed an isokinetic exercise protocol with and without beta-alanine supplementation. Muscle contractility was tested during the interset rest periods and during a 24 h recovery period. No significant effects were found, indicating that beta-alanine did not improve work output, fatigue, or muscle contractility.