A Placebo-Controlled Trial of Riboflavin for Enhancement of Ultramarathon Recovery

Post-exercise muscle pain and soreness have been well documented in ultramarathon running [1–8]; however, the precise etiology and pathophysiology remain elusive. Presumably, the process begins with mechanical damage to the muscle and connective tissue; is followed by inflammation, swelling, and the production of free radicals; and culminates in the pain and soreness felt during and after exercise [9–11]. Markers of inflammation and oxidative stress are known to be high following an ultramarathon [5, 7, 12–15].

Numerous pre- and post-exercise interventions using nutritional supplements and dietary strategies have been investigated for prevention or treatment of exercise-related muscle pain and soreness [10, 16]. Since the inflammatory response and free radical production with oxidative stress are likely involved in the mechanism leading to exercise-related muscle soreness, it seems plausible that supplementation with substances having anti-inflammatory or antioxidant properties may be an effective means of controlling such soreness. While research has provided some general support for the effectiveness of such substances in reducing exercise-related muscle soreness [10, 16], previous studies specific to ultramarathon running have demonstrated no effect. For instance, 6 weeks of vitamin E and C supplementation was found to have no effect on muscle damage, inflammatory markers, or muscle function recovery after a 50-km trail run [12, 13]. Furthermore, 3 weeks of supplementation with quercetin, another substance with known antioxidant properties, was not found to alter antioxidant capacity or oxidative damage, inflammation, muscle damage, or post-race muscle soreness from a 161-km ultramarathon [6, 15].

A common nutritional supplement that we believe has not been investigated for its effect on exercise-related muscle soreness is vitamin B2 (riboflavin). As for other flavonoids, riboflavin is known to exhibit antioxidant properties and protect tissue from oxidative damage [17–23]. Riboflavin is also important in cell repair and production and for protecting mitochondrial and other enzymes as a mitochondrial enzyme cofactor or cofactor precursor [24]. It is one of eight water-soluble B vitamins found in many foods and must be regularly supplied in the diet as it is not stored by the body [25]. Human trials have largely focused on the efficacy of riboflavin supplementation for migraine prophylaxis with favorable findings [26, 27]. Whether or not it might provide a protective role or enhanced recovery from exercise-induced skeletal muscle damage is unknown.

The purpose of this study was to investigate whether acute ingestion of riboflavin is effective at reducing muscle pain and soreness during and after completion of a 161-km ultramarathon, and in improving functional recovery after the event. The study was performed in association with the 161-km Western States Endurance Run (WSER) since this race is known to induce considerable muscle damage and pain [2, 3, 5–7, 28–32]. Based upon the evidence that riboflavin can protect tissue from oxidative damage and is important in cell repair, we hypothesized that riboflavin would be effective at reducing muscle pain and soreness and improving muscle recovery after this extreme level of exercise.

Of the 353 race entries, 44 runners enrolled in the study (22 in each group) and started the race. Of this group, 37 (84.1%, 20 in riboflavin group, 17 in placebo group) finished the race and 32 (18 in riboflavin group, 14 in placebo group) completed data collection. Of the 32 completing data collection, 8 had enrolled in advance of race registration and completed both pre-race 400-m run trials (5 in riboflavin group, 3 in placebo group). Overall race finish rate was 79.3% (280 of 353 starters), which was similar (p = .55) to that for the study participants.

The findings relative to muscle pain and soreness ratings for those subjects completing the study are shown in Fig. 1. Analysis of the data across all time points revealed no significant group (p = .14) or group by time interaction (p = .25), but there was a significant (p < .0001) time effect. Post-race values were statistically similar to pre-race values by post-race day 5. The pre-race and race data for all subjects providing data through 90 km (21 and 20 in riboflavin and placebo groups, respectively) were also analyzed. There was a significant group by time interaction (p = .023) and time (p < .0001) effect, but no group effect (p = .075), and post-testing revealed a significantly lower value (p < .01) for the riboflavin group than the placebo group at 90 km. This finding prompted further exploratory analysis of the data for those subjects completing the study with exclusion of the pre-race and post-race data (i.e., considering only the ratings during the race and at the finish) that yielded a significant (p = .043) group effect, as well as a significant (p < .0001) time effect, but no significant (p = .22) interaction effect.

The post-race 400-m run times are shown in Fig. 2. Significant time (p < .0001) and group (p = .016) effects were found, but no significant (p = .60) interaction effect was evident. Post-testing revealed significant group differences (p < .05) at post-race days 3 and 5. For the 8 subjects who had completed 400-m runs prior to the race, the post-race day 10 times were not statistically different (p = .10) from the pre-race times (mean ± SD, 82 ± 9, 80 ± 10, and 85 ± 7 s for pre-race trial 1, pre-race trial 2, and post-race day 10, respectively).

As shown in Table 1, pain medication use during the race was not different between groups but was at 50% among the riboflavin group and 36% among the placebo group. Considering the 9 subjects in the riboflavin group who reported using pain medication during the race, 7 used a NSAID and 2 used acetaminophen at a mean (range) of 29% (12–62%) of the maximum recommended 24-h dose. Of the 6 subjects in the placebo group who reported using pain medication during the race, 1 used a NSAID and 4 used either acetaminophen, paracetamol, or paracetamol with codeine at a mean (range) of 39% (10–75%) of the maximum recommended 24-h dose. Comparison of muscle pain and soreness ratings between subjects who used pain medication during the race with those not using pain medication during the race revealed no suggestion of a group or interaction effect, whether considering all time points (p = .43 and p = .61 for group and interaction effects, respectively) or just the ratings during the race and at the finish (p = .98 and p = .82 for group and interaction effects, respectively).

Subjects who received riboflavin correctly suspected they had received the treatment 50% of the time, whereas 23% of the placebo group incorrectly thought they were in the treatment group. These rates of suspicion about being in the treatment group were not statistically different (p = .16). There were 3 subjects in the riboflavin group and 1 subject in the placebo group who noted a yellow urine color.

This work is a preliminary examination of riboflavin for potential benefits of reducing muscle pain and soreness during and after strenuous exercise and at enhancing recovery from strenuous exercise. The findings suggest that the vitamin may have some benefits. Indeed, riboflavin supplementation immediately before and midway during prolonged exercise appeared to be linked with reduced muscle pain and soreness during and at the completion of the exercise, and there was some evidence for enhanced functional performance during the initial several days after the exercise. While the findings require cautious interpretation, they are adequately interesting to warrant further investigation.

Given that post-race plasma CK concentrations were similar between groups, there is no evidence from this work that riboflavin acts by reducing muscle cell rupture. Rather, it would seem that it must act by altering the physiological response to exercise in other manners. While the underlying mechanism of action cannot be established from this study, it seems conceivable that the antioxidant properties of riboflavin [17–23] could explain reduced muscle pain and soreness during exercise, although the lack of reduced muscle pain and soreness during recovery does not seem consistent with this mechanism. On the other hand, the mitochondrial protective function of riboflavin [24] might be a plausible explanation for the riboflavin group demonstrating enhanced functional recovery without improvement in muscle pain and soreness during the recovery period.

Participants of the WSER are generally well-trained and experienced ultramarathon runners given that a recent qualifying ultramarathon is required to gain entry into the race. In this regard, they were conditioned for this type of activity and were adapted for controlling and responding to significant muscle injury from prolonged running. It is possible that an effect of riboflavin could be even greater in a group of subjects who are more naïve to strenuous exercise.

We chose to provide two 100-mg doses of riboflavin which were received immediately prior to exercise and around 11–16 h later during the approximately 20–30 h it took to complete the race. This dose and schedule were chosen because we felt it would be feasible to achieve subject cooperation and that any effectiveness should still be evident even if this was not the optimal dose or dosing schedule. The doses we provided were well above the recommended dietary allowance for riboflavin of 1.3 mg/day for adult men and 1.1 mg/day for adult women [41]. While the body absorbs little riboflavin from single doses beyond 27 mg [42], the vitamin appears safe at much higher doses [41] and riboflavin supplements are typically available in 100-mg capsules with recommendations to take 1–2 per day. Plasma concentrations of riboflavin and flavocoenzymes have been shown to peak around 2 and 3.5 h, respectively, after a single 60-mg dose of riboflavin, but the plasma concentrations remain elevated for hours [42]. Considering this information, it is possible that a lower dose at more frequent intervals might be more effective and would be recommended for future studies of this nature if feasible in the study environment.

The present findings indicate that muscle pain and soreness ratings of our subjects had returned to pre-race levels by 5 days after the race. For the subsample of 8 subjects who performed the pre-race 400-m runs, their times at 10 days after the race were statistically similar to pre-race times, although mean times were still ~5% slower at 10 days after the race. Our prior work at the WSER had also demonstrated that muscle pain and soreness ratings had statistically returned to baseline by post-race day 5, but 400-m run times were not examined in that study beyond post-race day 5 at which time pre-race performance had not been fully recovered [3]. In the present work, we extended the post-race time period of examination to 10 days and found that this appears to be close to the timeframe of 400-m run recovery. This is not intended to suggest that athletes are fully recovered from a 161-km ultramarathon within 10 days or shortly thereafter but rather that our subjective measure of resting muscle pain and soreness and our objective measure of 400-m speed had nearly recovered during this relatively short time period.

The WSER serves as an excellent environment to induce muscle pain and damage as evident from prior work at the race [2, 3, 5–7, 28–32]. This is confirmed with the present work in which muscle pain and soreness ratings at the end of the race averaged ~7–8 on the 10-point scale (sore to very sore) and median post-race plasma CK concentrations were ~7000–13,000 U/L. However, subject recruitment is a challenge in performing research of this nature in a competition setting such as the WSER. This is reflected in the number of subjects we were able to recruit. In particular, it is unfortunate that the number of subjects completing the pre-race 400-m runs was so low, which limits the robustness of our interpretation of the findings relative to functional recovery. While the treatment and placebo groups appeared to be well matched and blinding appeared to be adequate in this study, we cannot be certain that the groups were well matched for baseline performance at the 400-m run.

We acknowledge some other limitations with this study resulting largely from constraints related to the study being performed at a competition. Perhaps most importantly is that a sizable percentage of the subjects (50 and 36% in the riboflavin and placebo groups, respectively) used pain medication during the race and some used pain medication in the 10-day post-race period. The use of NSAIDs during this event has been common, ranging from 32 to 57% among those participating in our prior research [3, 34, 43]. Interestingly, earlier work has demonstrated that NSAID use during the race was not effective at controlling post-race muscle soreness [4] though the effect of NSAIDs on muscle pain and soreness during the race has not been systematically examined. Among the present subjects using pain medication during the race, the usual dosage was relatively low compared with the maximal recommended dose during 24 h. Not surprisingly, our separate comparison of muscle pain and soreness ratings between subjects who used pain medication during the race and those not using pain medication during the race revealed no suggestion of an effect of the pain medication on this variable. Thus, given these considerations, it seems unlikely that the use of pain medication during the race confounded the present finding of lower muscle pain and soreness during and at the completion of the race among the riboflavin group compared with the placebo group. Another potential study limitation is that, because we did not assess dietary practices of the subjects, it is conceivable that one group had a greater intake of anti-oxidants than the other. Additionally, the 400-m runs were unsupervised and self-timed, but this was the most feasible approach and we believe this study population was capable of maximally exerting themselves during unsupervised trials and correctly recording the times. Finally, we also recognize that some might consider it ideal to have measured pre-race plasma CK concentrations and to examine the pre-race to post-race change in plasma CK concentration rather than just the post-race value. But, since these runners would have reduced training prior to the race, we would expect pre-race plasma CK concentrations to have been very low relative to the post-race values, as previously demonstrated [4–7], so not using the pre-race to post-race change would not have altered our interpretation of the findings for this variable.

From this work, we conclude that there is some evidence that riboflavin supplementation immediately before and midway through prolonged running may reduce muscle pain and soreness during and at the completion of the exercise and that there is some suggestion that riboflavin might enhance functional recovery after the exercise. We acknowledge that this study is a preliminary examination of riboflavin for this purpose and involved a small number of subjects in which the dose and dosing schedule might not have been optimal. As such, the findings appear intriguing and warrant additional investigation of riboflavin as a means to reduce muscle pain during exercise and to enhance post-exercise recovery.