A Placebo-Controlled Trial of Riboflavin for Enhancement of Ultramarathon Recovery
Hoffman et al. Sports Medicine - Open
A Placebo-Controlled Trial of Riboflavin for Enhancement of Ultramarathon Recovery
Martin D. Hoffman 0 1
Taylor R. Valentino 2
Kristin J. Stuempfle 4
Brandon V. Hassid 3
0 Ultra Sports Science Foundation , El Dorado Hills, CA , USA
1 Department of Physical Medicine & Rehabilitation, Department of Veterans Affairs, Northern California Health Care System , Sacramento, CA , USA
2 Department of Kinesiology, San Francisco State University , San Francisco, CA , USA
3 School of Medicine, University of Maryland , Baltimore, MD , USA
4 Health Sciences Department, Gettysburg College , Gettysburg, PA , USA
Background: Riboflavin is known to protect tissue from oxidative damage but, to our knowledge, has not been explored as a means to control exercise-related muscle soreness. This study investigated whether acute ingestion of riboflavin reduces muscle pain and soreness during and after completion of a 161-km ultramarathon and improves functional recovery after the event. Methods: In this double-blind, placebo-controlled trial, participants of the 2016 161-km Western States Endurance Run were assigned to receive a riboflavin or placebo capsule shortly before the race start and when reaching 90 km. Capsules contained either 100 mg of riboflavin or 95 mg of maltodextrin and 5 mg of 10% ß-carotene. Subjects provided muscle pain and soreness ratings before, during, and immediately after the race and for the 10 subsequent days. Subjects also completed 400-m runs at maximum speed on days 3, 5, and 10 after the race. Results: For the 32 (18 in the riboflavin group, 14 in the placebo group) race finishers completing the study, muscle pain and soreness ratings during and immediately after the race were found to be significantly lower (p = .043) for the riboflavin group. Analysis of the 400-m run times also showed significantly faster (p < .05) times for the riboflavin group than the placebo group at post-race days 3 and 5. Both groups showed that muscle pain and soreness had returned to pre-race levels by 5 days after the race and that 400-m run times had returned to pre-race performance levels by 10 days after the race. Conclusions: This preliminary work suggests that riboflavin supplementation before and during prolonged running might reduce muscle pain and soreness during and at the completion of the exercise and may enhance early functional recovery after the exercise.
Creatine kinase; Muscle fatigue; Muscle pain; Muscle soreness; Running
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
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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
exerciserelated 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 . 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 . 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.
Study Design and Subjects
This double-blind, placebo-controlled trial was performed
at the 2016 WSER, a 161.3-km ultramarathon through the
Sierra Nevada Mountains of Northern California. The
course is mostly single-track trail with 5500 m of
cumulative climb and 7000 m of cumulative descent. Other race
details have been provided elsewhere [33–36]. Nearby
weather station ambient temperatures during the race
ranged from a low of 1 °C at the start to a high of 34 °C.
The San Francisco State University Institutional Review
Board provided approval for the research with electronic
consent obtained during online enrollment and formal
consent obtained at race registration. Study participants
completing the pre-race data collection were provided a
t-shirt, and those completing the entire study were
provided a $50 (USD) gift certificate.
Subject recruitment was by electronic notices sent to
all race entrants 59 and 19 days before the race. Because
of inadequate subject recruitment, additional subjects
were recruited during race registration held the day
before the race start.
All study participants were met during race registration
for formal consenting, additional pre-race data collection,
and review of expectations for participation. Subjects were
then seen for further intervention before and during the
race as outlined below. Immediately after race completion,
subjects were escorted 30 m to a tent adjacent to the
finish line where post-race data collection and a blood
draw were performed. During the subsequent 10 days,
the subjects recorded additional information that was
then returned to the investigators electronically or by
mail. They were sent an email the evening after the race
reminding them of the post-race data collection and
were provided an electronic copy of the data sheet at
Subjects were asked to avoid any use of pain or
nonsteroidal anti-inflammatory drugs (NSAIDs) during the
race. Additionally, subjects were asked to avoid use of pain
medications or NSAIDs, compression garments, massage,
electrical stimulation, and thermal modalities in the 10 days
following the race. To check compliance, the post-race
data form requested that they provide details about use of
pain medications or NSAIDs during or after the race or
any of the above interventions during the 10 days after the
race. Subjects were also asked which group they thought
they were in and why.
Subjects were assigned to the riboflavin or placebo
group in an alternating fashion based on the order of
arrival to meet with the research team at race registration,
with the on-site researchers and subjects being blinded
to the group assignment. The subjects were told that
they would receive either a placebo or an essential
vitamin with flavonoid properties, but no other details of
the supplement under study were provided until after
data analysis had been completed.
All subjects received a capsule 0.5–1 h before the start
of the race with a cup of water and were observed to
swallow the capsule. They were also provided and
observed to take another capsule at the 90-km aid station.
Capsules were prepared by the investigators using
riboflavin (Nature’s Way Products, Inc., Green Bay, WI),
maltodextrin (Now, Bloomingdale, IL), and 10%
ßcarotene powder (BulkSupplements.com, Henderson,
NV). The treatment group received capsules
containing 100 mg of riboflavin, and the control group
received capsules containing 95 mg of maltodextrin and
5 mg of 10% ß-carotene. Because riboflavin can cause
urine to appear bright or fluorescent yellow  and
might allow subjects to suspect they were in the treatment
group, the small amount of ß-carotene was added to the
placebo, as done in a prior placebo-controlled trial of
riboflavin , since it can also cause a similar urine color
change. The amount of ß-carotene in each capsule was
approximately half of that in a single large raw baby carrot
, so we recognize it was unlikely to cause marked
urine discoloration, but it did allow us to legitimately tell
the subjects that they might notice a discoloration of the
urine regardless of their group assignment. We also
recognized that ß-carotene is an antioxidant but felt
that it would have no recognizable antioxidant effect at
such a low dose.
The body weight of each subject was obtained at race
registration and immediately after finishing the race using
the same scale (Sunbeam Products, Inc., Health o meter,
model 349KLX, Boca Raton, FL) placed on a firm, level
surface. For each measurement, the runner was clothed in
running attire and shoes but had no other items on their
body or in their hands.
Plasma Creatine Kinase Concentration
Plasma creatine kinase (CK) concentration was determined
immediately post-race from a blood sample taken from
the antecubital vein, with subjects seated in the upright
position. The samples were centrifuged for 10 min at
3400 rpm within 10 min of collection and then stored
in a cooler until transported to and analyzed by a
clinical laboratory for plasma CK concentrations (Siemens
Aktiengesellschaft, Dimension EXL, Munich, Germany).
Subjective Measurement of Muscle Pain and Soreness
Runners were asked to rate their perceived lower-body
muscle pain and soreness according to a 10-point Likert
scale with anchors of 1 (no soreness), 2.5 (dull, vague
ache), 4 (slight soreness), 5.5 (more than slight soreness),
7 (sore), 8.5 (very sore), and 10 (unbearably sore). This
approach has been previously used at the WSER [2–7]
and elsewhere , and the values have been found to
correlate with plasma CK concentrations [3–5]. Ratings
were provided at race registration, during the race at the
48-, 90-, and 126-km checkpoints, at the finish prior to
the blood draw, and each morning of the 10 days
following the race after being up and moving around for
approximately 30 min.
A 400-m run at maximal speed was used as a functional
measurement, which we have previously used
successfully [2, 3]. Those subjects enrolled in advance of race
registration were asked to perform this test twice on
separate days during the 21 days before the race, and all
subjects were asked to perform the test on days 3, 5, and
10 after the race. Subjects were sent an electronic
reminder the night before each post-race 400-m run. This
test is functionally specific for running, but not
overwhelmingly long and painful or so short and intense that
it would be a high risk for inducing a strain injury. After
an adequate warm-up, subjects were instructed to
selftime the run, starting from a standing start. Subjects were
asked to use the inside lane of a running track, assuring
an accurate distance. In the event they did not have access
to a track, they were allowed to utilize a level section of
road that had been measured to the correct distance. They
were asked to use the same site for all pre-race and
postrace testing and under conditions without significant wind
or other environmental variations.
Comparison of treatment and control groups (age, sex,
finish time, percent change in body mass from registration
to immediately post-race, post-race plasma CK
concentration, pre-race 400-m run time, average weekly running
distance, highest weekly running distance, and longest
training run) were made using unpaired t tests and the
chi-square test. Main outcome variables (muscle pain and
soreness rating and 400-m run time) were compared
between groups with two-way (group × time) repeated
measure analysis of variance (ANOVA) and Bonferroni
post-tests. These data were tested for normality with the
D’Agostino-Pearson normality test. The 400-m run time
data were skewed and successfully normalized before
analysis with the reciprocal function (i.e., transformed value =
1/original value). Statistical significance was set at p < 0.05.
A priori sample size determination was performed
based on muscle pain and soreness ratings from
previous research at the WSER [2–4]. Using a level of
significance of p < 0.05, an expected common SD of 1.5 points,
and 80% power, the predicted minimum sample size to
determine a meaningful group difference of 1.5 points
was 13 per group. Recent research at the WSER has
shown attrition rate to be roughly 25–30% [2, 3, 40], so
we aimed to recruit a minimum of 17 subjects per
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.
Selected characteristics of the subjects completing the
study are shown in Table 1. None of the examined
characteristics differed between groups, including the
postrace plasma CK concentrations. Furthermore, race finish
rate (91 and 77% for the riboflavin and placebo groups,
respectively) and study completion rate (82 and 64% for
the riboflavin and placebo groups, respectively) did not
differ statistically (p = .4 and p = .3, respectively) between
groups. One subject in the placebo group failed to
receive the capsule at 90 km, and two subjects in the
riboflavin group reported emesis within an hour after taking
the capsule at 90 km. All other subjects received both
doses and were confirmed to not have had emesis during
Table 1 Selected characteristics of the two study groups
the hour after taking a capsule. Post-race exercise behavior
appeared comparable between groups, and during the first
2 days post-race, none of our subjects reported exercise
more taxing than running less than 2 km or some walking.
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,
Body mass change from
registration to post-race (%)
Used any pain medication
during the race (%)
Used any pain medication
in 10 days post-race (%)
−3.6 ± 2.1
−2.6 ± 2.0
Post-race plasma CK (U/L)
6804 (3536–24,592) 12,819 (7560–46,965)
Fig. 1 Mean lower-body muscle pain and soreness ratings for the 2
groups. *p = .043 for group comparison considering only the
duringrace and finish data. Error bars represent 1 SD and are shown only in
1 direction for clarity
Fig. 2 Mean 400-m run times for the 2 groups. *p < .05 for
posttesting group comparison. Error bars represent 1 SD and are shown
only in 1 direction for clarity
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  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 . While the body absorbs little
riboflavin from single doses beyond 27 mg , the vitamin
appears safe at much higher doses  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 .
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
prerace performance had not been fully recovered . 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  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
prerace 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
ANOVA: Analysis of variance; CK: Creatine kinase; NSAID: Nonsteroidal
antiinflammatory drug; SD: Standard deviation; WSER: Western States Endurance Run
This research could not have been accomplished without the help of
numerous volunteers. We thank Sutter Auburn Faith Hospital for laboratory
services; John Fors, NP, Tracy Beth Høeg, MD, PhD, and Sonja A. Wilkey, MD,
for phlebotomy services; and the following individuals for assistance with
data collection: Dr. Jeffrey A. Chan, Kelly Cronin, George Daniel Cross, Casey
Curl, Maria Gonzalez, Jasmine Magallanes, and Tiffany Anne Morales. The
contents reported here do not represent the views of the Department of
Veterans Affairs or the US Government.
The work was funded by the Western States Endurance Run Foundation and the
Rossi Family Foundation and is also the result of work supported with resources
and the use of facilities at the VA Northern California Health Care System.
Availability of Data and Materials
The results, and what they reflect, are discussed in detail in the manuscript.
For this trial, participant-level data will not be publicly posted, but the authors
welcome collaborators with whom data may be shared for education and
MDH and KJS conceived and designed the experiments. MDH, KJS, and TRV
secured funding for the research. TRV, KJS, and BVH performed the experiments.
MDH analyzed the data and prepared the manuscript. All authors discussed and
revised the manuscript and approved the final manuscript.
Ethics Approval and Consent to Participate
The study was approved by the Institutional Review Board of San Francisco
State University and was performed in accordance with the ethical standards
of the Declaration of Helsinki. All participants provided written informed
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