72 • Eccentric exercise and inflammation

Eccentric exercise-induced delayed-onset muscle
soreness and changes in markers of muscle damage
and inflammation
Kazue Kanda1, Kaoru Sugama1, Harumi Hayashida2, Jun Sakuma3,
Yasuo Kawakami3, Shigeki Miura4, Hiroshi Yoshioka4, Yuichi Mori4,
Katsuhiko Suzuki1,3

1 Cooperative Major in Advanced Health Science, Tokyo University ofAgriculture and Technology/ Waseda University, Japan.
Institute for Sport Sciences, Waseda University, Japan.
3 Faculty of Sport Sciences, Waseda University, Japan.
4 Mebiol Inc., Japan.
2

ABSTRACTThe purpose of this study was to determine the relationships among delayed-onset
muscle soreness (DOMS), muscle damage and inflammatory responses to eccentric exercise and investigate the underlying mechanisms. Nine healthy males performed one-leg calf-raise exercise with their right leg on a force plate. They performed 10 sets of 40 repetitions of exercise at 0.5 Hz by the load corresponding to
the half of their body weight, with a rest for 3 min between sets. DOMS was evaluated by a visual analogue scale (VAS). Blood and urine samples were collected
before and 2, 4, 24, 48, 72 and 96 h post-exercise. Blood samples were analyzed
for leucocyte differential counts and neutrophil functions (migratory activity andoxidative burst activity). We also determined a serum marker of muscle damage,
myoglobin (Mb), and plasma and urinary prostaglandin E2 as an algesic substance. As for the inflammatory mediators, plasma and urine were analyzed forcytokines (interleukin (IL)-1β, IL-1 receptor antagonist, IL-2, IL-4, IL-6, IL-8, IL10, IL-12p40, IL-12p70, tumour necrosis factor-α, interferon-γ, monocyte chemotactic protein-1, granulocyte colony-stimulating factor, macrophage colonystimulating factor, and granulocyte macrophage colony-stimulating factor), leucocyte activation markers (calprotectin and myeloperoxidase), and neutrophil
chemotactic factor complement 5a. All subjects reported muscle soreness on subsequent days and VAS peaked at 72 h after exercise. Serum Mb concentration significantly increased (p<0.05) at 72 h after exercise as compared with the preexercise values which was correlated with the increases in VAS at 72 h (r=0.73,
p<0.05). Circulating neutrophil count and migratory activity increased significantly (p<0.01, and p<0.05, respectively) at 4 h after exercise, whereas there
Address for correspondence:
Katsuhiko Suzuki, MD, PhD, Faculty of Sport Sciences, Waseda University,
2-579-15, Mikajima, Tokorozawa, Saitama 359-1192, Japan.
Tel: +81-4-2947-6898, Fax: +81-4-2947-6898, Email: katsu.suzu@waseda.jp
EIR 19 2013

Eccentric exercise and inflammation • 73
were no significant changes in the other plasma and urinary inflammatory mediators. These results suggest that neutrophils can be mobilized into the circulationand migrate to the muscle tissue several hours after the eccentric exercise. There
were also positive correlations between the exercise-induced increases in neutrophil migratory activity at 4 h and the increases in Mb at 48 h (r=0.67, p<0.05).
These findings suggest that neutrophil mobilization and migration after exercise
may be involved in the muscle damage and inflammatory processes.

Key words: delayed-onset muscle soreness, eccentric exercise, exercise-induced
muscle damage, inflammatory mediators, neutrophils

INTRODUCTION

Unaccustomed exercise with eccentric muscle contraction and exhaustive exercise cause muscle damage, inflammation, leakage of muscle proteins into the circulation and soreness on and several days after, which is called delayed-onset
muscle soreness (DOMS) (14, 21, 24, 25). Although a large number of
researchers have investigated the effects of exercise on muscle damage and
DOMS in humans, there were various results in inflammatory responses (13, 17,30), which may depend on exercise mode, intensity, and duration, as well as anindividual’s sex and age.

Exercise disrupts skeletal muscle ultrastructurally, resulting in leucocyte infiltration and release of myocellular proteins such as myoglobin (Mb) into the circulation (3, 24, 28). Systemically, marked neutrophilia with a left shift (6, 23), andenhanced capacity of neutrophils to produce reactive oxygen species (ROS) havebeen documented after endurance exercise (12, 23). Peripheral leucocyte count has
also been shown to increase several hours after eccentric exercise (4). Histologicalexamination of muscle biopsy in humans demonstrates leucocyte accumulation inmuscle tissues (10, 29). After muscle and connective tissue damage following
exercise, neutrophils are rapidly mobilized into the circulation, and soon migrate
and infiltrate into the damaged tissue and produce ROS. If neutrophil functions,especially ROS production are over activated, tissue damage may occur. Within 24
h, neutrophils are replaced by macrophages which are active inflammatory cells
that produce several pro-inflammatory cytokines, and promote removal of debris
and remodeling of muscle tissue (9, 29). Indeed, several researchers investigated
the inflammatory responses to eccentric exercise in humans (15, 19, 32). Peake etal. reported that changes in circulating leucocyte count after eccentric exercise are
dependent on the muscle groups, or the amount of muscle mass recruited during
eccentric exercise (16). Concerning changes in leucocyte receptor expression andoxidative burst activity, there were no clear effects of different types of eccentric
exercise, but they might be due to differences in the type of eccentric exercise (16).
Cytokines are proteins which regulate immune and inflammatory responses. They
are classified into pro-inflammatory cytokines which promote inflammation, antiinflammatory cytokines which inhibit inflammation, immunomodulatory
cytokines which control inflammation, multifunctional cytokines, chemokines
EIR 19 2013

74 • Eccentric exercise and inflammationand colony-stimulating factors. Some of these substances are induced remarkably
in plasma and urine following exhaustive endurance exercise (26). Other substances, involved in inflammatory responses, are neutrophil chemotactic factor
complement 5a (C5a) and prostaglandin E2 (PGE2). Although numerous studies
have investigated the effects of eccentric exercise on exercise-induced muscle
damage, changes in plasma cytokines and neutrophil activation, there are several
contradictory findings, partly due to 1) differences in the mode and intensity of
exercise, 2) a limited range of variables measured, and 3) unreliable methods ofneutrophil activity measurement.
Migration of neutrophils to the tissue microenvironment is the first step to evoke
local inflammation. The production of ROS, not only of superoxide (O2–) andhydrogen peroxide (H2O2), but also of MPO-dependent HOCl production of neutrophils, can be measured by luminol-dependent chemiluminescence (LmCL) (7,23, 24, 27). Various neutrophil functions, such as migration and ROS production,
could work as a dual-edged sword on both sides of host defence and tissue injury(23, 27). Thus, the balance between beneficial and harmful effects of neutrophil
functions should be properly assessed. The use of hydrogel made it possible tomimic the in vivo microenvironment of neutrophil infiltration into tissues and
LmCL can be detected through the transmissive gel (27). In this method, neutrophil
migratory activity and ROS producing activity can be measured without the alteration of neutrophil functions by separating process in the conventional methods (7).

The aims of this study were at first to clarify relationships among muscle soreness, muscle damage markers, circulating leucocyte dynamics and changes in
inflammatory mediators in blood and urine of which we tried to detect any possible changes. Especially, we applied a newly-developed measurement system ofneutrophil migratory activity and ROS-producing activity by use of ex vivohydrogel methodology with extracellular matrix to the investigation of the mechanisms of muscle damage (27).

METHODS

Subjects
Nine untrained healthy males participated in this study. Their mean (±SD) characteristics were as follows: age (24.8±1.3 yrs), body mass (62.3±6.3 kg), and height(1.72±0.05 m). At the time of the study, the subjects had not been involved in any
hard exercise or resistance training for at least two weeks before the exercise
bout, and were not taking any supplements, or participating in recovery strategies
such as massage, stretching, or cryotherapy. The subjects were instructed tomaintain their usual daily schedule during the experiment. The study protocol
was approved by the ethics committee of Waseda University, Japan, and the subjects provided their informed consent.

Experimental design
Subjects performed a calf-raise exercise, including repetitive eccentric muscle
contractions with their right leg on a force plate. The range of motion of the ankle
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Eccentric exercise and inflammation • 75
joint during the exercise was regulated from -20° (dorsiflexion position) to 15°(planter flexion position) using a goniometer (SG 110/A, Biometrics, Newport,UK) with its ends attached onto the skin over the tibia and calcaneus. They performed 10 sets of 40 repetitions of exercise at 0.5 Hz by the load corresponding tohalf of their body weight, with a rest for 3 min between sets. DOMS was ratedusing a visual analogue scale (VAS) that had a 100-mm line with “no pain” on
one end and “extremely sore” on the other. Blood and urine samples were collected before and 2, 4, 24, 48, 72 and 96 h after the exercise.

Blood and urine sampling and analyses
Approximately 12 ml of blood samples were drawn by a standard venipuncturetechnique from the antecubital vein. Blood samples were collected into serum separation tubes and vacutainers containing heparin and EDTA. A portion of whole
blood was used to measure haemoglobin, haematocrit and complete blood cellcounts using an automatic blood cell counter (PocH100i, Sysmex, Kobe, Japan).
The serum separation tubes were left to clot at room temperature for 30 min, andthe vacutainers containing EDTA for plasma separation were immediately centrifuged at 1000×G for 10 min. Serum and plasma samples were then removed andstored at –80°C for later analysis. Serum concentrations of creatinine (Cr) and Mb
were measured using an automated analyzer (Model 747-400, Hitachi, Tokyo,
Japan). Urine samples were centrifuged immediately at 1000×G for 10 min toremove sediments, and the supernatants were stored at –80°C for later analyses.
Urinary concentrations of Cr were measured using an automated analyzer (Model
747-400, Hitachi, Tokyo, Japan). Enzyme-linked immunosorbent assay (ELISA)
kits were used to measure the plasma and urine concentrations of interleukin (IL)1β, IL-1 receptor antagonist (IL-1ra), IL-6, IL-12p70, tumour necrosis factor-α(TNF-α), monocyte chemotactic protein-1 (MCP-1), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GMCSF), and macrophage colony-stimulating factor (M-CSF) (R&D Systems; Minneapolis, MN, USA), IL-2, IL-4, IL-8, IL-10, IL-12p40, interferon-γ (IFN-γ), andC5a (Becton Dickinson Biosciences; San Diego, CA, USA), and calprotectin andmyeloperoxidase (HyCult Biotechnology; Uden, the Netherland). ELISA measurements were performed according to the instructions for each ELISA kit using a
microplate reader (VERSAmax; Molecular Devices, Sunnyvale, CA, USA). Plasma and urine concentrations of PGE2 (ENZO Life Sciences Inc; Farminglale, NY,
USA) were measured using a chemiluminescent microplate reader (FLUOstar
OPTIMA, BMG LABTECH, Offenburg, Germany).

Preparation of peptide-bound temperature-responsive polymer (G-TRP)
Collagen peptide (24 g; SCP-5000; Nitta Gelatin Co., Osaka, Japan) was dissolved in 96 ml of distilled water at 37°C and followed by reaction with 3.26 g ofN-acryloylsuccinimide (Kokusan Kagaku, Tokyo, Japan) for 4 days at 37°C toobtain polymerizable collagen peptide. N-isopropylacrylamide (108.5 g; Kojin,
Tokyo, Japan) and n-butylmethacrylate (4.26 g; Wako Chemical, Osaka, Japan)
were dissolved in 600 ml of ethanol and then 123 g of the above aqueous solutionof polymerizable collagen peptide was added. Under nitrogen atmosphere, 1 mlof N,N,N’,N’-tetramethylethylenediamine (Wako Chemical, Osaka, Japan) and
10 ml of 10 wt% ammonium persulfate (Wako Chemical, Osaka, Japan) aqueous
EIR 19 2013

76 • Eccentric exercise and inflammation
solution were added to the mixed solution, and then reacted for 5 h at 4°C, maintaining the nitrogen atmosphere. After the reaction, 30 l of cold (4°C) distilledwater were added and the mixture was concentrated to 3 l using an ultrafiltration
membrane (molecular weight cut off 100,000) at 4°C. This dilution and concentration process was repeated 5 times in order to remove impurities and low molecular species. Lyophilization and sterilization of the final concentrated solution
gave 105 g of peptide-bound temperature-responsive polymer (G-TRP).

Preparation of scaffold-thermoreversible gelation polymer (S-TGP) gel
Under a clean-air laminar hood workbench, 0.5 g of G-TRP and 0.5 g of TGP(Mebiol gel; Mebiol Inc, Kanagawa, Japan) were dissolved in 16.7 ml of Hank’sbalanced salt solution (HBSS) at 4ºC for overnight, yielding a viscous transparent
S-TGP gel uniform liquid without any bubbles for use in the experiments (27).
Mebiol gel is a pure synthesized biocompatible copolymer composed of thermoresponsive polymer blocks and hydrophilic polymer blocks, characterized by
its temperature-dependent dynamic viscoelastic properties and used as a biocompatible scaffold for three-dimensional culture without any toxicity (22). S-TGP
gel is a peptide-bound thermoreversible gel formed by mixing G-TRP with the
Mebiol gel. It liquefies at low temperature, turns to gel immediately upon warming, and returns to liquid state again when cooled.

Neutrophil functions
Peripheral blood samples were drawn from subjects using 2 ml Na-heparin tubes(Venoject II, Terumo Co., Tokyo, Japan). The blood samples were mixed with 2.5
mM luminol (5-amino-2, 3-dihydro-1, 4-phthalazinedione; Sigma Aldrich, MO,
USA) at a ratio of 1:1. Then, 150 µl luminol-blood samples were layered on 50 µl
S-TGP gel prepared in a tube at 37°C, and was promptly measured by LmCL (relative light unit: RLU) using a luminometer (Gene Light 55; Microtec Co., Ltd,
Funabashi, Japan). The samples were incubated at 37°C, and the production of
ROS from neutrophils was monitored in a kinetic mode for 60 min. After measurement of LmCL at 60 min, luminol-blood samples were removed and the tubes
with 50 µl S-TGP gel in which neutrophils migrated were washed three times
with PBS warmed at 37°C. Then, the tubes with gel were cooled on ice, and 50 µl
Turk solution (Wako, Osaka, Japan) were added and mixed well. The liquidobtained in this way were set on the C-Chip (Disposable haemocytometer,
Neubauer improved, DHC-No.1, Digital Bio, Seoul), and the migratory cell number was counted under the microscope. Migrated neutrophil number was calculated by 20 times multiplication of the counted cell number.

Statistical analysis
Data were analyzed using two-way analysis of variance. When significant timeeffects were evident, multiple comparisons were analyzed with Bonferroni adjustment. Associations between data were analyzed with Pearson’s correlation coefficient (r). Statistical significance was set at p<0.05, and data were presented asmeans ± standard deviations (SD).

EIR 19 2013

Eccentric exercise and inflammation • 77

RESULTS

Delayed-onset muscle soreness and muscle damage markers
Muscle soreness developed on subsequent days after one-leg calf-raise exercise.
It increased significantly 48 h and peaked around 72 h after exercise comparedwith the pre-exercise values (p<0.01) (Figure 1). Muscle soreness after exercise
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Figure 1. Time course of changes in delayed-onset muscle soreness (DOMS) following thecalf-raise exercise as determined by a visual analogue scale (VAS).
Values: means±SD (n=9). Statistics: Two-way ANOVA. Post-hoc test: Bonferroni adjustment. **p<0.01: vs Pre.

decreased at 96 h from 72 h, but remained elevated compared with the pre-exercise values (p<0.01) (Figure 1). Concerning blood markers of muscle damage,
Mb concentration significantly increased at 72 h after exercise (p<0.05) as compared with the pre-exercise values (Figure 2).
Differential leucocyte count
Peripheral leucocyte counts significantly increased at 4 h after exercise, due to theincrease in neutrophils (p<0.01), and they returned to the pre-exercise values at      

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Figure 2. Changes in myoglobin (Mb) concentration following the calf-raise exercise. Values: means±SD (n=9). Statistics: Two-way ANOVA. Post-hoc test: Bonferroni adjustment.
*p<0.05: vs Pre.
EIR 19 2013

78 • Eccentric exercise and inflammation       

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Figure 3. Changes in peripheral leucocyte counts following the calf-raise exercise. Values:means±SD (n=9). Statistics: Two-way ANOVA. Post-hoc test: Bonferroni adjustment.
**p<0.01: vs Pre.

24 h after the exercise. Lymphocytes and other leucocytes showed no changes(Figure 3).

Neutrophil functions
Neutrophil migratory activity increased at 4 h after exercise (p<0.05) and ROS
producing activity showed a trend to significantly increase (p=0.07). They both
returned to the pre-exercise value at 24 h after exercise (Figure 4).

Inflammatory substances
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Figure 4. Changes in neutrophil migratory activity and producing activity of reactive oxygen
species (ROS) as determined by Luminol-dependent chemiluminescence (LmCL) following
the calf-raise exercise. Values: means±SD (n=9). Statistics: Two-way ANOVA. Post-hoc test:
Bonferroni adjustment. LmCL p=0.07: vs Pre, migratory neutrophil counts *p<0.05: vs Pre.
EIR 19 2013

Eccentric exercise and inflammation • 79      !  "


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Figure 5. Associations between muscle soreness as determined by visual analogue scale(VAS) at 72 h and value of myoglobin (Mb) at 72 h. Values: percent changes of peak values
vs Pre (n=9). Statistics: Pearson's correlation coefficient. VAS (Pre-72 h) vs Mb (Pre-72 h):r=0.73, p<0.05      !  "

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Figure 6. Associations between migratory neutrophil count at 4 h and myoglobin concentration at 48 h. Values: percent changes vs Pre (n=9). Statistics: Pearson's correlation coefficient. Migratory nuetrophil count (Pre-4 h) vs Mb (Pre-48 h) : r=0.67, p<0.05

matory cytokines, colony-stimulating factors, leucocyte activation markers, neutrophil chemotactic factor C5a and algesic substance PGE2 showed no significant
changes (Table 1).
Relationships between muscle soreness and muscle damage markers
We investigated the correlations between the peak VAS value of muscle soreness,and peak value of muscle damage marker, neutrophil dynamics and inflammatory
mediators (all percent changes). There was a positive correlation between the percent changes of VAS at 72 h and Mb concentration at 72 h (r=0.73, p<0.05) (Figure 5). There was also a positive correlation between the percent changes of
migrated neutrophil count at 4 h and Mb concentration at 48 h (r=0.67, p<0.05)(Figure 6).
EIR 19 2013

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DISCUSSION

The aims of this study were at first to investigate the associations among DOMS
caused by calf-raise exercise, muscle damage marker in peripheral blood, andinflammatory mediators. Muscle soreness appeared at 24 h after exercise andsubsequently developed from 48 h to 72 h (p<0.01), and decreased at 96 h
though still high above compared with pre-exercise (p<0.01). Generally, DOMS
appears from several hours or one day after exercise and peaks at two or threedays after exercise (1, 14, 16). This study confirmed that calf-raise exercise witheccentric contractions caused DOMS. As a marker of muscle damage, serum
Mb concentration significantly increased at 72 h after exercise compared withpre-exercise. Numerous studies have reported on muscle damage and muscle
soreness, however, they did not always occur simultaneously according to thedegree of muscle damage and muscle soreness (4, 5, 13, 14). In this study, weobserved the similar time course and Mb was closely correlated with muscle
soreness at 72 h after exercise (r=0.73, p<0.05). These results confirm that thepresent calf-raise exercise caused sufficient muscle damage for the purpose of
the present study.

Several studies report that circulating leucocyte and neutrophil count increase
within several hours after eccentric exercise, but that is dependent on the intensity, duration, and type of exercise and muscle mass (16, 20). Saxton et al. reported that circulating neutrophil count increased 1.48 fold (Pre:2.9±0.4, 4h:4.3±0.5)
at 4 h after low systemic stress (repeated eccentric muscle action) and caused
1.76 fold (Pre:2.9±0.3, 4h:5.1±0.4) increase in neutrophils at 4 h after high systemic stress (bench-stepping) (20). Gleeson et al. reported the circulating leucocyte number decreased two and three days after a bench-stepping exercise,although it increased immediately after and still more increased at 1 to 4 h (5).
We demonstrated that total leucocyte count increased by 31 % (p<0.01) and circulating neutrophil count by 44 % (p<0.05) above pre-exercise level, respectively. Neutrophilia returned to pre-exercise values at 24 h after exercise. Neutrophil
migratory activity was also significantly increased (p<0.05) at 4 h after exercise.
There have been several studies that investigated the neutrophil migration into
the muscle after the muscle-damaging exercise (0-6 h) (2, 3, 8, 9, 11, 33), but
only Fielding et al. demonstrated neutrophil accumulation in the muscle (3). This
could be partly attributable to the fact that biopsy samples can depict quite limited parts of the muscle tissue. Paulsen et al. investigated leucocyte accumulation
into the exercised-muscle using radiolabeled leucocytes by scintigraphy (15).
Although they measured high radioactivity in the exercised-muscle, large individual differences were observed. We demonstrated a positive correlation
between the increase in neutrophil migratory activity at 4 h and the increase in
Mb concentration at 48 h (r=0.67, p<0.05). This result shows the possibility thatneutrophil is involved in the muscle damage and the inflammatory processes.

ROS production from neutrophils migrated into the hydrogel likened to muscle
tissue was assessed with LmCL. There was a trend for this to be increased(p=0.07) at 4 h after exercise. Pizza et al. reported that neutrophil O2- production
was increased (p<0.05) at 4 h following one-arm eccentric exercise (18). Suzuki
EIR 19 2013

82 • Eccentric exercise and inflammation
et al. also observed increased neutrophil count with a left shift, enhanced spontaneous mobility and ROS production after 90-min bicycling, suggesting that mobilized neutrophils from the bone marrow reserve have higher activity (23). They
also reported that the increases in Mb values at post 3 h were correlated closelywith the raise in neutrophil count at post and LmCL response at post and 1 h post
90-min bicycling, suggesting that neutrophil mobilization and activation might
affect muscle damage (24). The result in this study was similar, but we could
demonstrate the enhanced neutrophil migration by use of newly-developed exvivo methodology in imitation of tissue damage.

Although DOMS, muscle damage marker, circulating neutrophil counts and theirfunctions changed significantly following the eccentric exercise, plasma and urinary inflammatory mediators which we investigated were not changed significantly. Uchida et al. reported increasing serum PGE2 following bench press exercise despite no changes in plasma IL-1β, IL-6 or TNF-α (30). Although we also
investigated plasma and urinary PGE2 concentrations, they did not change. Serum
PGE2 may be produced by leucocytes during the time left to removal of the fibrin
clot and blood cells where neutrophilia occurs after exercise that might affect thehigher values. These results demonstrated that the one-leg calf-raise exercise
caused local inflammation and that neutrophil mobilization and migration werethe most affected variables among the wide range of tested inflammatory mediators. However, further research is needed to clarify the mediators that mobilize
neutrophils into the circulation and substances which are produced by migrated
neutrophils and promote inflammation.
In conclusion, one-leg calf-raise exercise caused DOMS, muscle damage,increases in circulating neutrophil number and migratory activity withoutchanges in the other inflammatory mediators. Neutrophils may be involved in theearly stage of muscle damage and the inflammatory processes after eccentric
exercise.

ACKNOWLEDGEMENTS

The authors thank volunteers who participated in the study. Also, we are very
grateful for the technical support of Dr. Noriaki Kawanishi and Dr. Masaki Takahashi, and for the English editing of Dr. Cecilia Shing. This study was partly supported by grants from the Ministry of Education, Culture, Sports Science andTechnology of Japan, the Grant-in-Aid for the Global COE Program “Sport Science for the Promotion of Active Life” (2010-2011), the Scientific Research (A)23240097, (B) 21300217, and Challenging Exploratory Research (2012-2013)
24650410.

1.

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