nutrients
Article
Consumption of An Anthocyanin-Rich Antioxidant
Juice Accelerates Recovery of Running Economy and
Indirect Markers of Exercise-Induced Muscle Damage
Following Downhill Running
Leonardo C. R. Lima 1,2,3,4,*, Renan V. Barreto 1, Natália M. Bassan 1,2, Camila C. Greco 1
and Benedito S. Denadai 1
1
Human Performance Laboratory, São Paulo State University, Av. 24-A, 1515, Rio Claro, SP 13506-900, Brazil
2
Centro Universitário Hermínio Ometto, Av. Dr. Maximiliano Baruto, 500, Araras, SP 13607-339, Brazil
3
Centro Universitário Salesiano de São Paulo, R. Baronesa Geraldo de Resede, 330,
Campinas, SP 13075-270, Brazil
4
Centro Universitário UniMetrocamp, R. Dr. Sales de Oliveira, 1661, Campinas, SP 13035-500, Brazil
*
Correspondence: leonardocrlima@gmail.com
Received: 18 July 2019; Accepted: 6 August 2019; Published: 23 September 2019


Abstract: This study examined the eﬀects of anthocyanin-rich antioxidant juice (AJ) on the recovery
of exercise-induced muscle damage (EIMD) and the running economy (RE) following downhill
running (DHR). Thirty healthy young men were randomly divided into two blinded groups and
consumed either AJ or placebo (PLA) for nine days (240 mL twice-a-day). On day 5, the participants
from both groups ran downhill (−15%) for 30 min at 70% of their maximal oxygen uptake (VO2max)
speeds. The changes in RE (oxygen uptake (VO2) and perceived eﬀort (PE) during 5-min runs at
80%VO2max) and EIMD (isometric peak torque (IPT), muscle soreness (SOR) and serum creatine
kinase activity (CK)) were compared over time and between the groups on the 4 days following DHR.
VO2 and PE increased (p < 0.05) immediately following DHR for both groups and remained elevated
for PLA until 48h post-DHR while fully recovering 24 h post-DHR for AJ. SOR was greater (p < 0.05)
for PLA throughout the study. CK increased for both groups and was greater (p < 0.05) for PLA at
96 h post-DHR. IPT decreased for both groups but recovered faster for AJ (72 h) compared to PLA
(no full recovery). AJ accelerated recovery of RE and EIMD and should be used in speciﬁc contexts,
but not chronically.
Keywords: running economy; antioxidant supplementation; anthocyanins; exercise-induced muscle
damage; recovery; muscle soreness
1. Introduction
The running economy (RE) is an important predictor of performance in endurance events.
It is deﬁned as the amount of oxygen required to sustain running at a ﬁxed submaximal speed [1].
RE represents, therefore, how eﬃcient athletes are during running. Athletes with similar maximal
oxygen consumptions (VO2max) may present diﬀerent performances in long-distance runs due to the
diﬀerences in RE [2].
Several factors inﬂuence RE acutely and chronically. A relationship between RE and neuromuscular
aspects exists. A growing body of literature investigated the eﬀects of exercise-induced muscle damage
(EIMD) on parameters associated with RE [3–5]. EIMD occurs when muscle tissue is damaged following
strenuous exercise, leading to compromised force production capacity, muscle soreness and leakage of
intracellular proteins to the circulation [6].
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Downhill running (DHR) has been reported as a damaging activity due to the high volume of
eccentric contractions performed during the breaking phase of running associated with oxidative
stress produced in the muscle by prolonged mitochondrial activity [3,4,7]. Assumpção et al. [8] have
reviewed the literature and showed that DHR compromises RE as much as countermovement jumps
and heavy-load squatting exercises [8].
Anthocyanins are phenolic compounds found in dark-colored fruits that act as a pigment in
nature [9]. However, evidence suggests that anthocyanin rich foods have powerful antioxidant
and anti-inﬂammatory properties when consumed by humans [10]. In fact, there is evidence that
consuming anthocyanin-rich juices leads to faster recovery of markers of EIMD following resistance
training [11] and endurance events [12]. The authors have recently reviewed the literature on the
eﬀects of consuming tart cherry juice (which is rich in anthocyanins and other phenolic compounds)
in recovery from EIMD [13]. However, to the best of the authors’ knowledge, there are no studies
investigating if the consumption of anthocyanin-rich juice accelerates the recovery of RE following
damaging bouts.
The aim of the present study was to investigate the eﬀects of anthocyanin-rich juice consumption
on the magnitude of changes and time-course of recovery of markers of EIMD following a DHR bout.
Our hypothesis was that the consumption of an antioxidant juice rich in anthocyanins would promote
faster recovery of markers of EIMD and RE when compared to a placebo treatment.
2. Materials and Methods
2.1. Participants
Thirty healthy male physical education students (age: 22.3 ± 2.6 years; height: 176.6 ± 6.4 cm;
body mass 77.1 ± 10.5 kg) participated in the present study. The inclusion criteria for the present
study were: Aged between 18 and 30 years-old; not having any experience with strength or aerobic
training in the last six months; being a non-smoker and not having had lower-limb injuries in the
last six months. They were instructed to refrain from intense physical activity, to keep their regular
dietary habits and to drink plenty of water during the experimental period. All the participants read
and signed an informed consent prior to their participation in the study, which was approved by the
institution’s ethical board. A total of thirty participants were enrolled for the study and all of them
completed the experimental protocol (15 per group). All the interactions and procedures in the present
study were in accordance to the Declaration of Helsinki for research involving humans.
2.2. Experimental Design
The study was conducted under double-blind, placebo-controlled conditions. The participants
were allocated to either experimental (EXP) or placebo (PLA) groups in a randomized fashion.
Randomization was performed by the lead examiner using a draw application for smartphone.
Before being assigned to each group, the participants were familiarized to the experimental procedures.
The familiarization sessions included performing maximal isometric contractions on an isokinetic
dynamometer (System 3, Biodex Systems, Shirley, NY, USA). The participants also had the opportunity
to run for one minute on a treadmill (Pulsar, h/p/Cosmos, Germany) with 0% inclination to familiarize
with treadmill running. In the second familiarization visit, the participants’ VO2max were determined.
After at least ﬁve days following the last familiarization session, the participants ran downhill
(−15%) for 30 min at 70% of their VO2max speed. Chen et al. [3] showed that this protocol leads to
signiﬁcant damage to lower limb muscles and compromised RE. The knee extensors isometric peak
torque (IPT), and markers of RE (oxygen uptake (VO2) and perceived eﬀort (PE) during submaximal
running bouts) were assessed 15 min before, 15 min after, and 1–4 days following DHR. Lower limb
muscle soreness was assessed 15 min before and 1–4 days following DHR. Serum creatine kinase (CK)
activity was assessed 15 min before, 2 and 4 days following DHR.
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The participants in the EXP group consumed 240 mL of an anthocyanin-rich antioxidant juice
(Antiox, Juxx, Brazil) twice a day with a 12 h interval between doses at the day of the DHR bout and
on the 4 days preceding and 4 days following it. Participants in the PLA group consumed a placebo
consisting of water mixed with maltodextrin. The antioxidant juice and the placebo solution were
isocaloric (106 Kcal per dose), isovolumetric (240 mL) and had the same amount of carbohydrates
per dose (26 g). The experiment was conducted in a double-blinded fashion with all subjects and
examiners blinded for the treatment being administered. The treatment bottles were opaque and the
participants from both groups did not have contact with each other to avoid cross-contamination.
The experimental design is illustrated in Figure 1.
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The participants in the EXP group consumed 240 mL of an anthocyanin-rich antioxidant juice 
(Antiox, Juxx, Brazil) twice a day with a 12 h interval between doses at the day of the DHR bout and 
on the 4 days preceding and 4 days following it. Participants in the PLA group consumed a placebo 
consisting of water mixed with maltodextrin. The antioxidant juice and the placebo solution were 
isocaloric (106 Kcal per dose), isovolumetric (240 mL) and had the same amount of carbohydrates per 
dose (26 g). The experiment was conducted in a double-blinded fashion with all subjects and 
examiners blinded for the treatment being administered. The treatment bottles were opaque and the 
participants from both groups did not have contact with each other to avoid cross-contamination. 
The experimental design is illustrated in Figure 1. 
 
Figure 1. The experimental design of the study. The biggest dash represents downhill running. AJ: 
antioxidant juice; PLA: placebo; CK: creatine kinase. 
2.3. Antioxidant Juice 
The participants in the EXP group consumed an anthocyanin-rich antioxidant juice that 
consisted of a mixture of clarified apple juice with prum, blueberry, maquiberry, raspberry and 
cranberry concentrates. Each dose of the juice (240 mL) contained 58 mg of anthocyanins and an 
antioxidant capacity of 67,680 μmoL/mL of Trolox equivalent, as assessed by the oxygen radical 
absorbance capacity (ORAC5) scale to identify antioxidant capacity. The evidence suggests that 
consuming tart cherry juice with equivalent levels of anthocyanins attenuates muscle soreness and 
accelerates the recovery of muscle function following damaging bouts [11,12,14]. The daily intake 
and timing of antioxidant juice consumption in the present study were planned based on previous 
studies that showed enhanced recovery of indirect markers of EIMD due to the consumption of 
similar anthocyanin-rich juices [11,12,15,16]. 
2.4. Maximal Oxygen Uptake 
VO2max was determined through a treadmill incremental test. The participants ran on a 
treadmill (Pulsar, h/p/Cosmos, Nussdorf-Traunstein, Germany) wearing a mask attached to a breath-
by-breath gas analyzer (Quark PFT Ergo, Cosmed, Pavona, Italy) that recorded oxygen uptake (VO2) 
and carbon dioxide (CO2) production during exercise until test cessation. The incremental test started 
with a three-minute warm-up at 7 km/h followed by continuous 1 km/h increments every minute. 
The treadmill inclination was constant and set at 1%. The criteria adopted for cessation of the test 
were: (1) Heart rate of 95% of the predicted maximal (220-age); (2) the respiratory exchange ratio 
greater than 1,15; (3) voluntary fatigue. Following filtering of the data, VO2max was considered as 
the greatest VO2 value recorded and sustained for at least 15 s during the test. The speed at which 
VO2max was reached (sVO2max) was also recorded. 
Figure 1. The experimental design of the study. The biggest dash represents downhill running. AJ:
antioxidant juice; PLA: placebo; CK: creatine kinase.
2.3. Antioxidant Juice
The participants in the EXP group consumed an anthocyanin-rich antioxidant juice that consisted
of a mixture of clariﬁed apple juice with prum, blueberry, maquiberry, raspberry and cranberry
concentrates. Each dose of the juice (240 mL) contained 58 mg of anthocyanins and an antioxidant
capacity of 67,680 µmoL/mL of Trolox equivalent, as assessed by the oxygen radical absorbance
capacity (ORAC5) scale to identify antioxidant capacity. The evidence suggests that consuming tart
cherry juice with equivalent levels of anthocyanins attenuates muscle soreness and accelerates the
recovery of muscle function following damaging bouts [11,12,14]. The daily intake and timing of
antioxidant juice consumption in the present study were planned based on previous studies that showed
enhanced recovery of indirect markers of EIMD due to the consumption of similar anthocyanin-rich
juices [11,12,15,16].
2.4. Maximal Oxygen Uptake
VO2max was determined through a treadmill incremental test. The participants ran on a treadmill
(Pulsar, h/p/Cosmos, Nussdorf-Traunstein, Germany) wearing a mask attached to a breath-by-breath
gas analyzer (Quark PFT Ergo, Cosmed, Pavona, Italy) that recorded oxygen uptake (VO2) and
carbon dioxide (CO2) production during exercise until test cessation. The incremental test started
with a three-minute warm-up at 7 km/h followed by continuous 1 km/h increments every minute.
The treadmill inclination was constant and set at 1%. The criteria adopted for cessation of the test were:
(1) Heart rate of 95% of the predicted maximal (220-age); (2) the respiratory exchange ratio greater than
1,15; (3) voluntary fatigue. Following ﬁltering of the data, VO2max was considered as the greatest
VO2 value recorded and sustained for at least 15 s during the test. The speed at which VO2max was
reached (sVO2max) was also recorded.
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2.5. Running Economy
The running economy was assessed at a ﬁxed-speed of 5 min runs at 80% of individual sVO2max.
This intensity was chosen based on the ﬁndings of Chen et al. [4] that RE at 80% and 90% sVO2max,
but not at 70% sVO2max, is compromised following DHR. Therefore, this study adopted the lowest
intensity at each RE which is compromised following DHR. The breath-to-breath gas exchanges were
registered during RE tests and the mean VO2 was recorded during the ﬁfth minute of each test. At the
end of each RE test, the participants rated their perceived eﬀort in a scale that varied from 6 to 20 [17].
The VO2 at the last minute of the RE tests and the perception of eﬀort were recorded as metabolic and
perceptual indices of RE, respectively.
2.6. Indirect Markers of Exercise-Induced Muscle Damage
IPT, muscle soreness and serum CK activity were assessed as indirect markers of EIMD. To assess
IPT, the participants performed two 5 s maximal voluntary isometric contractions in an isokinetic
dynamometer with a 180 s recovery interval between contractions. A signal acquisition device with
a sampling frequency of 1000 Hz (Miotool, 200/400, Miotec, Porto Alegre, Brazil) was synchronized
with the dynamometer (System 3, Biodex Systems, Shirley, NY, USA) during the maximal voluntary
isometric contractions for greater precision during data acquisition. The participants were seated at the
dynamometer following the manufacturer’s guidelines, with their trunks, hips and dominant thighs
ﬁrmly secured to the chair, their knees ﬂexed at 70◦, and their legs ﬁrmly attached to the dynamometer
shaft. They were instructed to perform knee extensions as quickly and forcefully as possible during 5 s
with strong verbal encouragement being provided by the examiners. The acquired data was saved and
stored for subsequent analyses.
The data obtained during the maximal voluntary isometric contractions was ﬁltered (Butterworth
ﬁlter, low pass, 4th order, with a 15 Hz cut-oﬀ frequency) and analyzed in a MatLab environment
(MatLab 6.5, Mathworks, Natick, MA, USA). IPT was considered as the greatest value in the torque-time
curve. The contraction with the greatest IPT was used for further analyses.
Muscle soreness was quantiﬁed using a 1000 mm visual analogs scale with the saying “not sore at
all” and “very, very sore” at the extremities. The participants were instructed to rate their perceived
soreness after climbing up and down from a 45 cm chair with their dominant limb without external
assistance. They performed this test following 5 min of seated rest and could perform as many
repetitions as necessary. They were instructed to mark the visual analogs scale according to the soreness
they felt on their knee extensors after completing the stepping exercise.
Serum CK activity was quantiﬁed by spectrophotometric analyses. Further, 500 µL of blood was
extracted from the participant’s earlobes 5 min after the application of a vasodilator ointment (Finalgon,
Pharma GmbH & Co. KG, Aachen, Germany) to avoid hemolysis. The blood samples were allowed to
clot for 10 min and centrifuged for 10 min at 56,000 rpm (Microhemato, Modelo 2410, Fanem, São Paulo,
Brazil) and the serum samples were extracted and stored at −70 ◦C for further analyses. Serum CK
activity was determined using a commercial kit (CK-NAC UV, Wiener Lab, Rosário, Argentina) and a
spectrophotometer (Bio-2000, Bioplus, São Paulo, Brazil). The reference values for healthy men for the
method used ranged between 24 and 195 U/l.
2.7. Statistics
Data normality was conﬁrmed using the Shapiro-Wilk test. Data sphericity and homogeneity
were conﬁrmed using the Mauchly and Levene tests, respectively. The diﬀerences between groups in
baseline values for all dependent variables as well as anthropometric data were tested by the student’s
t-test. The changes over time and between groups were compared using the mixed model ANOVAs
for repeated (time) and non-repeated (groups) measures with Bonferroni post-hoc tests. All analyses
were performed in a professional software (Statistical Package for Social Sciences 17, IBM, Armonk,
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NY, USA). The signiﬁcance levels were set at p <0.05. The data are expressed as the means ± standard
deviation unless otherwise stated.
3. Results
The participants’ mean age, body mass, height, body mass index, VO2max, sVO2max, DHR speeds
and RE-test speeds are presented in Table 1. No signiﬁcant diﬀerences in such variables were found.
Table 1. The characteristics of the sample for the experimental (EXP) and placebo (PLA) groups.
PLA (n = 15)
EXP (n = 15)
Age (years)
22.8 ± 2.8
21.9 ± 2.3
Body mass (kg)
79.5 ± 11.8
74.6 ± 8.7
Height (m)
1.74 ± 0.07
1.77 ± 0.06
BMI (kg/m2)
26.2 ± 3.2
23.7 ± 2.2
VO2max (mL/kg/min)
41.8 ± 5.7
43.7 ± 4.3
sVO2max (km/h)
13.9 ± 1.4
14.7 ± 1.2
Downhill Running Speed (km/h)
9.7 ± 1
10.3 ± 0.9
Running Economy Test Speed (km/h)
10.1 ± 1.2
10.5 ± 1
PLA: Placebo group; EXP: Experimental group; BMI: Body mass index; VO2max: Maximal oxygen uptake; sVO2max:
Speed at which the maximal oxygen uptake was achieved.
No signiﬁcant diﬀerences between groups were found for baseline values of VO2 (CON:
33 ± 3.8 mL·kg−1·min−1; EXP: 35.1 ± 3.3 mL·kg−1·min−1), perceived eﬀort (CON: 11.5 ± 1.2; EXP:
12.3 ± 1.2), IPT (CON: 290 ± 34 Nm; EXP: 278 ± 36 Nm), knee extensor muscle soreness (CON: 0 ± 0 mm;
EXP: 0 ± 0 mm) and serum CK activity (CON: 106 ± 49 U.l−1; EXP: 126 ± 40 U.l−1). The signiﬁcant group
versus the time interactions were found for VO2 (F(5) = 20.05, p < 0.01), perceived eﬀort (F(5) = 4.86,
p < 0.01), IPT (F(5) = 3.80, p = 0.003), knee extensor muscle soreness (F(4) = 3.82, p < 0.01) and serum
CK activity (F(2) = 3.70, p = 0.31).
The pairwise comparisons showed that VO2 signiﬁcantly increased for both groups immediately
following DHR and fully recovered 24 h and 72 h post-exercise for the experimental and control groups,
respectively. VO2 was signiﬁcantly greater for the control group than the experimental group 24 h
post-DHR. This was also the case for perceived eﬀort. The absolute changes in VO2 and perceived
eﬀort over time following DHR are presented in Figure 2.
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Figure 2. Cont.
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Figure 2. The absolute changes in oxygen uptake (A) and perceived effort (B) over time following 
downhill running. * p < 0.05 compared to the baseline values in the same group. ‡ p < 0.05 compared 
to the experimental group at the same time-point. CON: control group; EXP: experimental group. 
The isometric peak torque significantly decreased (p < 0.05) for both groups immediately after 
DHR and remained so throughout the entire experimental period for the control group, but fully 
recovered 72 h post-DHR for the experimental group (Figure 3). 
Figure 2. The absolute changes in oxygen uptake (A) and perceived eﬀort (B) over time following
downhill running. * p < 0.05 compared to the baseline values in the same group. ‡ p < 0.05 compared to
the experimental group at the same time-point. CON: control group; EXP: experimental group.
The isometric peak torque signiﬁcantly decreased (p < 0.05) for both groups immediately after
DHR and remained so throughout the entire experimental period for the control group, but fully
recovered 72 h post-DHR for the experimental group (Figure 3).
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Figure 3. The relative changes in isometric peak torque over time following downhill running. * p 
<0.05 compared to the baseline values in the same group. CON: control group; EXP: experimental 
group. 
Knee extensor muscle soreness significantly increased (p < 0.05) 24 h following DHR and 
remained so during the whole experiment for both groups. However, knee extensor muscle soreness 
was significantly (p < 0.05) greater for the control group at all time-points. Serum CK activity 
significantly increased 48 h following DHR and remained elevated until 96 h following DHR for both 
groups. Serum CK activity was significantly (p < 0.05) greater for the control group at 96 h post-DHR. 
The changes in knee extensor muscle soreness and serum CK activity are presented in Figure 4. 
Figure 3. The relative changes in isometric peak torque over time following downhill running. * p <0.05
compared to the baseline values in the same group. CON: control group; EXP: experimental group.
Knee extensor muscle soreness signiﬁcantly increased (p < 0.05) 24 h following DHR and remained
so during the whole experiment for both groups. However, knee extensor muscle soreness was
signiﬁcantly (p < 0.05) greater for the control group at all time-points. Serum CK activity signiﬁcantly
increased 48 h following DHR and remained elevated until 96 h following DHR for both groups. Serum
CK activity was signiﬁcantly (p < 0.05) greater for the control group at 96 h post-DHR. The changes in
knee extensor muscle soreness and serum CK activity are presented in Figure 4.
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Figure 4. The changes in knee extensor muscle soreness (A) and serum creatine kinase (CK) activity 
(B) over time following downhill running. * p <0.05 compared to baseline values in the same group; ‡ 
p <0.05 compared to the experimental group at the same time-point. CON: control group; EXP: 
experimental group. 
4. Discussion 
The aim of the present study was to investigate the acute impact of antioxidant juice 
consumption on changes in RE and the recovery of indirect markers of EIMD following DHR. It was 
hypothesized that phenolic compounds present in the antioxidant juice—especially anthocyanins—
would accelerate recovery of muscle soreness, serum CK activity, muscle function and RE following 
DHR, and this was confirmed by the obtained data. 
The data from the control group showed that DHR significantly compromises RE at 80% 
sVO2max—measured as VO2 and the perceived effort—with full recovery reached 3 days following 
the exercise bout. DHR also led to significant changes in the indirect markers of EIMD (IPT, muscle 
soreness and serum CK activity) with full recovery not being reached within four days following the 
exercise bout for the control group. These findings corroborate what has been previously reported in 
the literature and confirm that recovery kinetics are different between RE and the indirect markers of 
EIMD [8]. 
Figure 4. The changes in knee extensor muscle soreness (A) and serum creatine kinase (CK) activity
(B) over time following downhill running.
* p <0.05 compared to baseline values in the same
group; ‡ p <0.05 compared to the experimental group at the same time-point. CON: control group;
EXP: experimental group.
4. Discussion
The aim of the present study was to investigate the acute impact of antioxidant juice consumption
on changes in RE and the recovery of indirect markers of EIMD following DHR. It was hypothesized
that phenolic compounds present in the antioxidant juice—especially anthocyanins—would accelerate
recovery of muscle soreness, serum CK activity, muscle function and RE following DHR, and this was
conﬁrmed by the obtained data.
The data from the control group showed that DHR signiﬁcantly compromises RE at 80%
sVO2max—measured as VO2 and the perceived eﬀort—with full recovery reached 3 days following
the exercise bout. DHR also led to signiﬁcant changes in the indirect markers of EIMD (IPT, muscle
soreness and serum CK activity) with full recovery not being reached within four days following the
exercise bout for the control group. These ﬁndings corroborate what has been previously reported in
the literature and conﬁrm that recovery kinetics are diﬀerent between RE and the indirect markers
of EIMD [8].
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The present study found that antioxidant juice consumption results in faster recovery of muscle
function as well as attenuated muscle soreness and serum CK activity following an exercise bout
consisting of 30 min of DHR. Our ﬁndings are similar to those which showed that consuming tart cherry
(Prunus cerasus L.) juice accelerates recovery of the indirect markers of EIMD following the diﬀerent
types of exercise bouts (i.e., resistance exercise training, maximal isokinetic eccentric contractions,
DHR, marathon running and stochastic cycling) [11,12,16,18,19].
However, little is known about the mechanisms underlying downhill running-induced muscle
damage. It is generally accepted that EIMD is characterized by two distinct events. In the ﬁrst event,
the mechanical strain imposed by unaccustomed exercise damages the sarcolemma and ultrastructural
sarcomere proteins [20]. This results in compromised muscle function due to the disrupted contractile
and structural proteins as well as the compromised excitation-contraction coupling [6]. The mechanical
damaging event is usually aggravated when eccentric contractions are performed during unaccustomed
exercise bouts due to the unique motor unit recruitment patterns during such contractions [21].
The mechanical damage is followed by cellular signaling for repair, which triggers an inﬂammatory
response consisting of the migration of neutrophils and monocytes (which diﬀerentiate into
macrophages once in the damaged site) [22]. The immune cells promote the degradation of cellular
debris through phagocytosis by producing oxygen reactive species. However, this degradation is
not exclusive to cellular debris, but also aﬀects healthy, functioning, structures of adjacent myocytes.
This is referred to as the second event of EIMD and leads to muscle soreness, increased CK release
to the blood stream and, possibly, further loss of muscle function [13]. It is yet to be determined if
the oxidative stress produced by the mitochondrial respiratory chain during DHR anticipates and/or
aggravates the second event of EIMD. It is, however, well established that DHR signiﬁcantly aﬀects the
indirect markers of EIMD [8,23].
The data obtained in the present study suggests that muscle function was significantly compromised
immediately following DHR for both groups. This was expected, since the consumption of antioxidant
juice is not expected to strengthen the sarcolemma nor impact the motor unit recruitment patterns,
attenuating the ﬁrst EIMD event. However, accelerated recovery kinetics were observed for both
RE (VO2 and perceived eﬀort) and muscle function (IPT) (Figures 2 and 3). The perceived eﬀort
and VO2 fully recovered two days earlier for the experimental group with signiﬁcant diﬀerences
between the groups observed 1 day following DHR. Similarly, IPT reached full recovery during the
study in the experimental group while it remained compromised throughout the entire study for the
control group. No signiﬁcant diﬀerences were found between the groups for the IPT values. Although
previous studies have reported attenuated changes in IPT following damaging bouts when associated
with consumption of tart cherry juice [11], accelerated recovery kinetics are also important when
investigating strategies to attenuate EIMD [16,24].
The diﬀerences between changes in RE and muscle function observed among the groups in the
present study might be explained by the antioxidant properties of anthocyanins in the antioxidant
juice. Previous studies showed that anthocynin-rich tart cherry juice reduced total oxidative stress and
circulating levels of C-reactive protein other oxygen reactive species [12,16,25]. It has been reported that
anthocyanins (as well as other phenolic compounds) scavenge free radicals secreted by lymphocytes
and produced in the mitochondrial respiratory chain [26].
It has also been reported that consuming foods as rich in anthocyanins as the antioxidant juice used
in the present study decreases circulating levels of pro-inﬂammatory cytokines such as interleukin-6
and tumor necrosis factor-α following damaging bouts [12,19], potentially attenuating the second event
of EIMD. This might also explain the attenuated muscle soreness for the experimental group observed
at all assessment points in our study. The delayed-onset muscle soreness is frequently described as a
symptom of the inﬂammation that occurs in the muscle and fascia following eccentric-biased activities
due to the interaction of algesic pro-inﬂammatory substances such as histamines, bradykinins and
prostaglandins with nociceptors [27]. Hence, if a treatment attenuates inﬂammation, it also attenuates
the ensuing soreness caused by it.
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Serum CK activity increased for both groups following downhill running, peaking 4 days after
it. However, serum CK activity was greater for the control group at its peak. As an intracellular
enzyme, the increased CK activity in the bloodstream is a sign of membrane and tissue damage [28].
Peak CK activity in the bloodstream occurs later than other indirect markers of EIMD since it must
be transported from the lymph to the circulation [28]. Signiﬁcantly greater serum CK activity for the
control group suggests either greater mechanical stress (which was not the case, since both groups
exercised at identical volumes and intensities) or that membrane damage caused by lipolytic enzymes
such as phospholipase A2—which is activated by pro-inﬂammatory cytokines [29]—was greater in
the absence of the treatment investigated in the present study. It can, therefore, be assumed that the
anti-inﬂammatory properties of the treatment investigated in the present study attenuated secondary
damage to healthy myocytes induced by inﬂammation.
Although oxidative-stress or inﬂammatory markers were not assessed in the present study, it is
important to notice that the concentration of anthocyanins in the antioxidant juiced consumed by the
participants in the experimental group is similar to those of cherry juices used in studies that found
attenuated inﬂammatory responses and total oxidative status. Associated with the observation of
similar eﬀects on the indirect markers of EIMD between our treatment and those previously reported,
this adds to the presented rationale.
To the best of the authors’ knowledge, no previous study investigated the impact of consuming
antioxidant/anti-inﬂammatory treatments in the magnitude of changes and recovery kinetics of RE
following damaging bouts. This study found that not only does consuming antioxidant juice accelerate
recovery of RE markers, but it also attenuates changes 1 day following DHR. In fact, our results suggest
that RE is only compromised immediately following DHR when consuming antioxidant juice. This has
important implications regarding training protocols and, especially, competitive schedules.
In speciﬁc contexts, athletes are submitted to short-term competitions at which they are expected
to perform in subsequent days. In such contexts, it is important for endurance athletes to maintain
their eﬃciency, and EIMD from previous days might be an issue. Training camps are also an example
of condensed endurance events during which it is of the best interest of athletes to perform as well
as possible. Our ﬁndings indicate that consuming an antioxidant juice rich in anthocyanins might
be a good strategy to maintain eﬃciency and attenuate muscle soreness in such contexts. Caution is
warranted when transferring the ﬁnding of the present study to trained athletes. The participants in our
study presented VO2max values between 40−45 mL·kg−1·min−1, which are not compatible with trained
athletes. The evidence suggests that antioxidant status is greater for athletes compared to sedentary
controls [30]. Hence, there is a possibility that additional antioxidant properties of anthocyanin-rich
foods do not further improve the already-high antioxidant response to damaging exercises in trained
athletes. However, Howatson et al. [12] showed that the consumption of Prunus cersasus L. accelerates
recovery of muscle function and soreness following marathon running in experienced runners. Further
studies are warranted to investigate if anthocyanin-rich foods accelerate recovery of RE in elite athletes.
The continuous use of antioxidant juices during endurance training programs should not be
encouraged. The evidence suggests that, despite the beneﬁcial eﬀects of antioxidant supplementation
in the recovery from EIMD and RE, oxidative stress might be an important component for training
adaptation [31]. Merry and Ristow [32] reviewed the literature on this topic and concluded that a
balance in redox signaling is the key to optimal endurance adaptation and long-term antioxidant
supplementation can blunt the physiological stress imposed by exercise, consequently compromising
optimal training adaptation. This should be taken into consideration when planning nutritional
strategies for training and competitions.
The assessment of oxidative status and markers of inﬂammation is important, and the absence of
these variables is a limitation of our study. While this study did ﬁnd a signiﬁcant impact of the treatment
in our main outcome, the direct markers of secondary damage to better elucidate the mechanisms
of faster RE recovery associated with antioxidant juice consumption were not assessed. The authors
encourage further investigation of such mechanisms. This study also did not carry out dietary analyses.
Nutrients 2019, 11, 2274
10 of 12
This might have implications regarding an already high antioxidant status due to the high anthocyanin
intake by the participants prior to the experiment. Since this study only recommended the participants
to maintain their regular dietary habits but did not control nor assess them during the experimental
period, this might also be considered a limitation of the present study. Another limitation of the
present study is the intensity at which RE was assessed. Future studies should assess the impacts
of antioxidant juice consumption on the changes in RE at diﬀerent intensities following DHR and
other damaging bouts. Finally, there seems to be a lack of standardization for the composition of
antioxidant products investigated in the literature. this study focused on anthocyanin concentration
and antioxidant capacity when choosing our treatment, as well as the commercial availability in our
country. Therefore, the ﬁndings presented in the present study represent the impact of this speciﬁc
antioxidant juice on changes in RE and the indirect markers of EIMD, which may not be the same when
using antioxidant juices with other compositions. It is recommended that practitioners and colleagues
focus on anthocyanin concentration when choosing which juice to prescribe/investigate.
5. Conclusions
In conclusion, this study shows that consuming an anthocyanin-rich antioxidant juice four days
prior to, at the day and four days following DHR resulted in the accelerated recovery of RE and
muscle function as well as attenuated muscle soreness. These data suggest that this nutritional strategy
might be useful to maintain satisfactory performance in condensed competitions and training camps.
Caution is warranted when planning long-term antioxidant supplementation, as training adaptations
might be blunted. Future studies are needed to clarify the mechanisms underlying faster recovery of
RE when consuming antioxidant juice.
Author Contributions: Conceptualization, L.C.R.L. and B.S.D.; data curation, L.C.R.L., R.V.B. and N.M.B.; formal
analysis, C.C.G. and B.S.D.; funding acquisition, L.C.R.L. and B.S.D.; investigation, L.C.R.L., R.V.B. and N.M.B.;
methodology, L.C.R.L. and B.S.D.; project administration, L.C.R.L. and B.S.D.; resources, L.C.R.L.; Supervision,
C.C.G. and B.S.D.; writing–original draft, L.C.R.L. and R.V.B.; writing–review & editing, N.M.B, C.C.G. and B.S.D.
Funding: This work was funded by the São Paulo Research Foundation (FAPESP) grant number 2013/23585-4 and
the APC was funded by the São Paulo Research Foundation (FAPESP) grant number 2019/17202-1.
Acknowledgments: The authors would like to thank Professor Glyn Howatson for his contributions to this work
and all participants for their eﬀort.
Conﬂicts of Interest: The authors declare no conﬂicts of interest.
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Consumption of An Anthocyanin-Rich Antioxidant Juice Accelerates Recovery of Running Economy and Indirect Markers of Exercise-Induced Muscle Damage Following Downhill Running.
Lima, Leonardo C R,Barreto, Renan V,Bassan, Natália M,Greco, Camila C,Denadai, Benedito S