The Effects of L-Citrulline and Malic Acid on Substrate Utilisation and Lactate Elimination

Abstract

Endurance athletes often aim to improve their aerobic metabolism. The aim of this pilot study was to examine if malic acid and L-citrulline supplementation can improve aerobic metabolism and lactate elimination. Nine young (23.9 ± 1.9 years) recreational male athletes participated in this study. Following a standardised breakfast and a body composition analysis (InBody720), 6000 mg of citrulline and 3000 mg of malic acid or a placebo of 300 mL of water were consumed on three separate days in a cross-over design using a double-blind method. Sixty minutes after the supplementation, participants completed a ramp bicycle spiroergometer protocol (35 W/3 min) until reaching a respiratory exchange ratio (RER) of 1.1, followed by a 9 min active recovery. Cadence, heart rate (HR), rate of perceived exertion (RPE), respiratory parameters and lactate levels were registered. The RPExHR value was calculated to accurately characterise exhaustion. During the exercise protocol, citrulline supplementation induced significantly lower RER values at 70-105-140 W compared to malic acid and the placebo, respectively. There was no difference in lactate levels neither during rest nor at RER 1.1. RPExHR rate values were significantly lower after malic acid supplementation compared to placebo at 175 and 210 W. Power at RER 1.1 was higher after malic acid (+4 W) and citrulline (+5 W) supplementation. Although the supplementation failed to decrease lactate levels, lower RER and RPE values may indicate a performance-enhancing benefit.

1. Introduction

Endurance athletes often aim to achieve a more efficient aerobic metabolism and lactate-eliminating mechanisms to maintain exercise performance at higher intensities.

Both citrulline and malic acid can be found in their natural form in foods we eat every day. Watermelon is known for its high citrulline content, whereas apples and cherries contain large amounts of malic acid [1,2,3]. They are also popular supplements that are taken by people suffering from kidney diseases with the goal of increasing blood flow thus treating hypertension and erectile dysfunction [4]. With regard to sporting performance, these supplements are mostly used blended together as a citrulline-malate 2:1 powder which resembles the basis of many pre-workout supplements, used to increase blood flow to the working muscle, thus enhancing muscle contraction [5,6].

The citric acid cycle plays an important role in aerobic metabolism, as every aerobic pathway produces acetyl-CoA, which enters this cycle and becomes further metabolised. Malate is an intermediate product of the citric acid cycle which is created from fumarate by the enzyme fumarase and is further converted to oxalacetate by the enzyme malate dehydrogenase [7]. Malate can also be produced directly from pyruvate via an anaplerotic reaction [8]. Exogenous malate may improve citric acid cycle function, which might lead to a more efficient aerobic metabolism [8].

L-citrulline has many important roles in the human body, two of which are worth highlighting. Firstly, citrulline is a urea cycle intermediate that is responsible for the excretion of ammonia, which is a byproduct of amino-acid breakdown [7]. Secondly, citrulline plays an important role in vasodilation where l-arginine is converted to N-hydroxyl-arginine and then into citrulline by the nitric-oxide enzyme in the endothelium of the vascular wall. This process is accompanied by nitrogen monoxide exiting into the smooth muscle tissue which leads to muscle relaxation [7]. Therefore, citrulline may provide better blood flow, possibly enhancing lactate elimination [7].

There is a substantial amount of research on this topic. However, in most of these studies, citrulline or malic acid is not given in isolation but in the form of citrulline–malate, which contains citrulline and malic acid in a 2:1 ratio [5,6]. Therefore, these studies are unable to answer the question of whether the isolated administration of these supplements can benefit sporting performance. Furthermore, most of these studies examine the effects of citrulline–malate on resistance training, not aerobic endurance [5,6]. Nonetheless, these studies show significant performance-enhancing benefits, including an increase in repetitions performed until failure, lower blood lactate levels and a decreased rate of perceived exertion (RPE) [5,6].

Far more studies have been conducted involving only citrulline [9,10,11] than malic acid [12] supplementation regarding sporting performance. However, research on L-citrulline supplementation provides contradictory results. Most research papers confirm the theory that citrulline supplementation increases plasma nitrate levels [13]; however, this does not always lead to an increase in performance. Bailey et al. (2016) observed that although plasma citrulline and arginine levels were higher after supplementation, this was accompanied by higher resting blood pressure and did not improve exercise time until exhaustion [14]. In those studies, which show benefits for aerobic performance, the effects were comparable to the effects on resistance training: increased time until exhaustion (e.g., swimming until exhaustion), lower lactate and ammonia levels and decreased RPE at the same intensity [9,10]. These benefits occurred in response to long-term supplementation rather than acute ingestion [9,15].

The research examining the possible sporting performance-enhancing effects of isolated, acute malic acid ingestion is even more uncertain. Having said that, acute malic acid supplementation may indeed be beneficial since, in an animal model, mice improved their swimming times until exhaustion [12]. The reviewed literature [9,10,11,12] clearly suggests that L-citrulline and malic acid can decrease RER and lactate values and an athlete’s perceived exertion, hence enhancing sports performance.

The RER value is used to describe an individual’s respiratory exchange ratio. It can be measured using a gas exchange analyser, which measures the oxygen-to-carbon-dioxide ratio in the inhaled and exhaled air. This ratio enables us to draw conclusions regarding an individual’s substrate utilisation [16,17]. During exercise, the human body must catch up with the increased demand for ATP by the muscles which can be produced primarily from carbohydrates and fat but also from amino acids. When the intensity is increasing, less fatty acids can be metabolised as they can only be broken down under aerobic conditions. Therefore, the ratio of carbohydrates to fatty acids metabolised shifts to a higher amount of carbohydrates which increases the RER value. The usual resting RER value of 0.8 can increase up to 1.2–1.3 during maximal effort [18].

The RPE (rate of perceived exertion) is a subjective value that is used in sports science to measure an individual’s perception of exhaustion [19,20]. The most well-known RPE scale is the Borg scale [19] where 6 indicates no effort and 20 indicates maximal effort.

The RPExHR( heart rate) value is the arithmetical product of the subjective RPE and the objective HR. Combining HRwith the RPE may give a more accurate picture of an individual’s state of fatigue under a given load [21].

The aims of this study were to investigate the possible benefits of citrulline as well as malic acid supplementation on aerobic endurance. In order to clarify the often-contradictory results and quantify the possible performance-enhancing benefits of separate citrulline and malic acid supplementation, we focused on measuring RER values and blood lactate levels to gain insight into an athlete’s metabolic response to the ingested supplements. Furthermore, we wanted to obtain a comprehensive picture of how the participant’s perception of fatigue changed after supplementation; thus, we decided to record RPE values throughout the experiment and to use the subjective RPE rating combined with objective HR data (RPExHR to characterise the participant’s sense of exhaustion.

Based on the literature on this topic we hypothesised the following:

(1)

L-citrulline supplementation will significantly decrease blood lactate levels during exercise;

(2)

L-citrulline supplementation will significantly decrease RER values during exercise;

(3)

L-citrulline and malic acid supplementation will significantly decrease RPE.

2. Materials and Methods

2.1. Participants

Nine healthy, young (age: 23.9 ± 1.9 years, body height: 181.9 ± 4.7 cm, body mass: 75.8 ± 5.0 kg) recreational male athletes (training 3-5x per week, VO2 max.: 2851.0 ± 426.4 mL/min) participated in this pilot study. We excluded athletes who were younger than 19 or older than 27 years old, who did not pass our medical examination (resting electrocardiogram [ECG], blood pressure measurement), trained less than 3 or more than 5 times a week, were taking medication/supplements or suffered from any chronic or acute disease or injury at the time. This study was conducted in accordance with the Declaration of Helsinki for Human Research. The protocol was approved by the Ethics Committee Board of the Hungarian University of Sports Science (TE-KEB/10/2022). In order to ensure the participants’ safety, a questionnaire was used which was developed for medical examinations by the Hungarian Sports Clinic (https://www.osei.hu/parizs-2024.html?id=139, accessed on 10 October 2022). Participants received verbal and written information about the protocol. Once familiarised with the study protocol, participants were asked to sign a consent form. Participants were allowed to withdraw from the study at any time without giving a reason. In addition, we composed a standard breakfast (

Table 1. Composition of the standard breakfast consumed by the participants.

	Energy (kcal)	Protein (g)	Carbohydrates (g)	Fat (g)
2 pieces of buns	305	10	63	0
7 slices of salami	285	16	1	24
1 tomato	22	1	2	0
Total	612	27	66	24

), which they consumed exactly 120 min prior to the body composition measurement, and we also asked the participants to rest in the evenings prior to their test days. When preparing the breakfast, we took into consideration the energy requirements of the exercise test protocol and the appropriate ratio of macronutrients, adjusted to sex and age, using the same brands every time. Data management was conducted anonymously. All measurements were carried out in the buildings of the Hungarian University for Sport Science, Department of Health Sciences and Sport Medicine.

2.2. Supplementation and Dosages

Participants completed the protocol three separate times on three separate days at the same time of the day with 48 h of recovery in between. The supplementation was either 6000 mg of L-citrulline + 300 mL of water, 3000 mg of malic acid + 300 mL of water or a placebo + 300 mL of water. No additional fluid was given until after the second body composition test at the end of the protocol. The supplements were taken in the same capsule form and the placebo was prepared by pouring out the capsule’s content. When choosing dosages, we wanted to keep the 2:1 ratio of citrulline–malate but also opted for a lower dosage, as citrulline–malate ingestion in higher doses can lead to gastrointestinal distress [22]; therefore, we agreed on a dose of 6000 mg. This was a double-blind cross-over protocol where participants were tested during rest, exercise and during active recovery.

2.3. Anthropological Measurements and Resting Protocol

Exactly 120 min after the participants consumed their standard breakfast, we measured their body composition via InBody 720 (Biospace Co., Ltd., Seoul, Republic of Korea) to ensure almost equal hydration levels between our participants during the protocol, followed by a medical examination which included measuring ECG for 5 min with a 12-lead machine (MSB CEP DB, 2010) and blood pressure (Omron, 2012) in seated position, taking the average of three measurements. Afterwards, the participants laid down on a medical bed, and we installed the gas exchange analyser and recorded their physiological parameters for 15 min with the aim of providing the participant sufficient time for psychological adaptation to the situation and stabilisation of their resting physiological processes. The ingestion of the supplements took place with the given dosages during the resting protocol’s 16–20 min, and participants continued to stay in a lying position for an additional 35 min until the supplements taken were absorbed. HR (Polar H-10 chest strap), lactate from the earlobe (Lactate Plus Sport Meter—Nova Biomedical, Waltham, MA, USA) and energy expenditure (kcal/min), as well as other respiratory parameters such as RER, BF (breathing frequency), V‘CO2 (carbon dioxide production), V’O2 (oxygen uptake), by the gas exchange analyser, were recorded (Jaeger CPX Vyntus, SentrySuite Version 2.17 software). After 55 min in a supine position, the participants transferred to the bicycle ergometer (Monark Ergomedic 839E) where they completed the exercise protocol.

2.4. Exercise Protocol

During the exercise protocol, the participants started to pedal with a power of zero watts (W), which was increased incrementally by 35 W every three min [17]. Three minutes at a given power equals one increment. We decided to start at such a low resistance of zero W because most participants had not used a bicycle ergometer before and were not trained cyclists. We also wanted to investigate whether there are effects of L-citrulline and malic acid at very low intensities. RER, HR and other respiratory parameters were recorded constantly during the whole exercise protocol, which we complemented by capturing pedalling cadence every minute as well as RPE values every third minute via the Borg scale (6 to 20), with it being the last minute of each increment. We found it important to record pedalling cadence as a higher cadence usually leads to a slightly higher HR [23]. In order to ensure as identical as possible conditions for each test, participants were asked to hold the same cadence throughout the whole exercise protocol as on their first test day.

The resistance progressively increased until the test person reached an RER value of 1.1 and maintained it for 15 s when the test personnel stopped the protocol. We chose this RER value because it is a high enough intensity where a significant amount of lactate builds up in the muscles but the participant’s current psychological state (e.g., pain threshold, monotony tolerance, etc.) does not affect the outcome with respect to maximal power/increments completed [24] (Figure 1b).

2.5. Active Recovery Protocol

After completing the exercise protocol, we continued to take measurements during a 9 min long active recovery, where the test person pedalled at a resistance of 0 W. RER, HR and other respiratory parameters were recorded continuously. At the 1st, 3rd, 5th, 7th and 9th minute, the test personnel performed lactate testing via blood from the earlobe, and participants gave their RPE estimates. Immediately after the 9 min long active recovery protocol, we repeated the body composition, ECG and blood pressure measurements in order to track any possible changes in body fluids or abnormal changes in the cardiovascular system.

2.6. Statistical Analysis

Due to the limited sample size, the impact of supplementations on the studied indicators during the rest period and exercise was assessed using non-parametric Friedman’s analysis of variance (ANOVA). Effect sizes were estimated by Kendall’s coefficient of concordance. The coefficient was interpreted as follows: 0.1 ≤ 0.3: small effect; 0.3 ≤ 0.5: moderate effect; and ≥0.5: strong effect [25]. To determine significant differences between treatments, a pairwise Wilcoxon signed-rank test with Bonferroni correction was conducted as a post hoc analysis. Exact p-values were employed to determine the significance level. The statistical analyses were performed using SPSS v. 23, and the hypotheses were tested at a 5% level of significance.

3. Results

3.1. RER and Lactate Levels before Supplementation

Friedman ANOVA tests did not reveal significant differences in RER and lactate values before the supplementation (Figure 2).

3.2. RER and RPExHR during Exercise

Friedman ANOVA tests revealed significant differences in RER values during the exercise protocol at 70 W (χ² = 6.889; N = 9; p < 0.05; Kendall’s W = 0.383), 105 W (χ² = 10.667; N = 9; p < 0.05; Kendall’s W = 0.593) and 140 W (χ² = 8.222; N = 9; p < 0.05; Kendall’s W = 0.457). Additionally, Wilcoxon pairwise comparisons revealed that citrulline significantly reduced RER values at powers of 70 W, 105 W and 140 W compared to malic acid (Wilcoxon, Z = −2.31, N = 9, Bonferroni-corrected p < 0.0167; Z = −2.67, N = 9, Bonferroni-corrected p < 0.0167; and Z = −2.67, N = 9, Bonferroni-corrected p < 0.0167) and compared to the placebo (Wilcoxon, Z = −2.19, N = 9, Bonferroni-corrected p < 0.0167; Z = −2.43, N = 9, Bonferroni-corrected p < 0.0167; and Z = −2.31, N = 9, Bonferroni-corrected p < 0.0167) (Figure 3). Absolute mean and standard deviation values are shown in

Table 2. Mean and standard deviation (±SD) of RER values at different loads during exercise.

	Placebo RER	Malic Acid RER	Citrulline RER
70 W	0.857 ± 0.087	0.860 ± 0.064	0.820 ± 0.075
105 W	0.927 ± 0.088	0.928 ± 0.069	0.898 ± 0.085
140 W	0.970 ± 0.075	0.972 ± 0.067	0.931 ± 0.083

.

Friedman ANOVA tests revealed significant differences in RPExHR values during the exercise protocol at powers of 175 W (χ² = 6.00; N = 7; p < 0.05; Kendall’s W = 0.429) and 210 W (χ² = 6.348; N = 6; p < 0.05; Kendall’s W = 0.529). Additionally, Wilcoxon pairwise comparisons revealed that malic acid significantly reduced RPExHR values at powers of 175 W and 210 W compared to the placebo (Wilcoxon, Z = −2.36, N = 7, Bonferroni-corrected p < 0.0167; and Z = −2.37, N = 6, Bonferroni-corrected p < 0.0167) (Figure 4). Absolute mean and standard deviation values are shown in

Table 3. Mean and standard deviation (±SD) of RPExHR values at different loads during exercise (malic acid–placebo).

	Placebo RPExHR	Malic Acid RPExHR
175 W	1825.1 ± 511.48	1633.5 ± 497.8
210 W	2082.3 ± 638.71	1953.4 ± 687

.

Table 4. The averageHR values of three different supplementation groups during exercise.

	Rest End	0 W	35 W	70 W	105 W	140 W	175 W	210 W	Peak Load
Placebo	75.5 ± 12.5	76.9 ± 11.4	85.5 ± 11.4	100.3 ± 12.3	117.2 ± 16.2	130.1 ± 16.5	142.0 ± 17.7	154.3 ± 19.4	160.0 ± 12.0
Malic acid	70.4 ± 7.5	75.1 ± 9.2	84.2 ± 8.9	98.2 ± 9.7	113.3 ± 12.6	126.7 ± 13.7	141.4 ± 17.2	151.7 ± 18.4	158.3 ± 12.4
Citrulline	70.9 ± 9.6	74.5 ± 9.9	84.4 ± 11.4	98.5 ± 10.0	114.0 ± 14.9	128.1 ± 16.1	141.6 ± 16.9	152.4 ± 19.9	161.6 ± 16.9

and

Table 5. The average RPE values of three different supplementation groups during exercise.

	Rest End	0 W	35 W	70 W	105 W	140 W	175 W	210 W	Peak Load
Placebo	8.0 ± 1.8	7.2 ± 1.2	7.9 ± 1.5	9.1 ± 2.1	10.0 ± 2.4	11.4 ± 2.7	12.9 ± 3.1	13.5 ± 3.2	13.6 ± 2.5
Malic acid	7.8 ± 1.3	7.3 ± 1.2	7.5 ± 1.2	8.4 ± 1.3	9.6 ± 1.3	10.9 ± 2.0	11.6 ± 2.7	12.9 ± 3.5	13.8 ± 3.1
Citrulline	7.0 ± 0.9	6.9 ± 1.2	7.3 ± 1.4	8.4 ± 1.5	9.2 ± 1.8	10.8 ± 1.9	12.1 ± 2.6	12.3 ± 2.1	13.8 ± 1.8

summarise the average HR and RPE values of three different supplementation groups during exercise.

3.3. RER and Lactate Levels during Active Recovery

Malic acid and citrulline supplementation had no effect on lactate levels at maximum power (Figure 5).

Neither malic acid nor citrulline supplementation had a significant effect on RER, RPE and lactate values during the 9 min active recovery protocol (Figure 6). Time points of lactate, RER and RPE measurements during active recovery are shown on the x-axis. RER (left y-axis) and lactate (right y-axis) values are displayed accordingly.

4. Discussion

The present study aimed to answer the question of whether the separate and acute administration of L-citrulline and malic acid could improve aerobic endurance. Our results show that L-citrulline significantly lowered RER values at 70, 105 and 140 W, while malic acid significantly decreased RPExHR values at 175 and 210 W. On the other hand, neither of the supplements affected lactate levels or increased maximal power at RER 1.1 significantly. There are multiple explanations for the findings presented above. Although Kohara et al., (2011) who studied a swimming until exhaustion test in mice [10], and Eroglu et al., (2017) who monitored a group of handball players during a 4-week training block [15], found lower lactate levels after citrulline supplementation, in the present study, neither citrulline nor malic acid had a meaningful impact on lactate levels. As we used similar dosages to other studies and malic acid also did not impact lactate levels on its own, there may be an interaction between L-citrulline and malic acid which led to lower blood lactate levels in the research where they used a citrulline–malate blend [15]. Therefore, further research is needed to identify the pathway through which malic acid may highlight L-citrulline and its impact on blood lactate levels [15].

Kobayashi et al. (2016) observed an increase in performance during a cycling time trial after supplementing with citrulline for 7 days [10], while Huang et al. (2007) administered malic acid for 30 days to mice which significantly increased their swimming time until exhaustion [12]. Nonetheless, we expected to see a higher power output at RER 1.1 compared to the placebo in our participants. Although the differences in maximal power did not reach statistical significance, supplementing with these two compounds may actually be worth considering for professional athletes. For competitions at the highest level, where placings are often decided by a few seconds or even less, a 4–5 Watt increase in power is certainly beneficial to every athlete. Considering the low cost and accessibility of these supplements, athletes who wish to improve their aerobic endurance may consider using these supplements.

According to Jeukendrup (2011), the most common reasons for fatigue during long-distance events are dehydration, hyponatremia and the depletion of glycogen stores [26]. One way to prevent glycogen depletion during exercise is by utilising higher amounts of fatty acids. Dokladny et al. (2018) identified the range for maximal fat oxidation (MFO) to be 45–65% of one’s VO2max [27]. When further increasing intensity, the 70:30 ratio of fatty acid/carbohydrate metabolism starts shifting more towards the breakdown of additional glucose [28]. Our results seem to suggest that supplementing with L-citrulline as well as malic acid may decrease RER values at low-to-medium intensities which would enable the athlete to supply more of their energy demands from fatty acids and be less reliant on glucose metabolism, thus sparing glycogen stores. This may apply mostly to long-distance events, where the depletion of glycogen can be a major factor in a drop in performance (long-distance triathlons, marathons, ultramarathons, etc.).

Vandewalle et al. (2018) made an observation that there may be a discrepancy between RPE, HRand lactate values at given intensities [29]. They concluded that, in order to determine an athlete’s actual state of fatigue, it is necessary to incorporate RPE besides HRand/or lactate as people often perceive a given load differently. The significantly lower RPExHRvalues observed following malic acid supplementation can be useful to reduce perceived fatigue during training and competition. Furthermore, we encourage coaches and self-coached athletes to use this metric as it interlinks objective and subjective parameters which helps to estimate the athlete’s current state of fatigue and how they perceive a certain training load.

5. Conclusions

This study showed no significant effects of L-citrulline and malic acid supplementation on aerobic performance. There was no significant metabolic benefit as L-citrulline did not decrease blood lactate levels at rest, at RER 1.1 or during active recovery. However, L-citrulline significantly decreased RER values at low-to-medium intensities, while malic acid decreased RPExHR values at moderate intensities. Therefore, these supplements might be beneficial for endurance athletes, especially in delaying the onset of glycogen depletion and preventing fatigue. The present research is limited to a small but homogenous sample. Further understanding of how these two supplements interact in endurance-trained athletes is required.

Strengths and Limitations

In the present study, we aimed to fill some gaps in the current research about citrulline and malic acid supplementation with the main focus being the effect on aerobic endurance with separate administration of said supplements. As we were striving to ensure that the participants were as similar as possible, we had to sacrifice the size of the sample for its homogeneity, especially with regard to the participants’ age and sex. Conducting studies with more participants may reveal thus far unknown performance-enhancing benefits to citrulline and malic acid supplementation.