ORIGINAL RESEARCH
Preventive Effects of 10-Day Supplementation With Saffron
and Indomethacin on the Delayed-Onset Muscle Soreness
Abbas Meamarbashi, PhD and Ali Rajabi, MSc
Objective: Delayed-onset muscle soreness (DOMS) often occurs
after unaccustomed eccentric exercise and reduces exercise perfor-
mance. We aimed to study the preventive effects of saffron and
indomethacin on the biochemical and functional indicators of DOMS
after 1-session eccentric exercise.
Design: A 10-day, randomized, double-blind, placebo-controlled,
pretest–posttest design.
Setting: Controlled research laboratory.
Participants: Thirty-nine nonactive male university students
randomly divided into saffron (n = 12), indomethacin (n = 12),
and control (n = 15) groups.
Interventions: Saffron group received 1 capsule containing dried
saffron powder (n = 12, 300 mg/d), indomethacin group received 75
mg indomethacin (n = 12, 25 mg thrice a day), and control group
(n = 15) received placebo capsules, 1 week before and 3 days after
eccentric exercise. Ten days before and 24, 48, and 72 hours after
muscle soreness protocol, the maximum isometric and isotonic
forces, plasma creatine kinase (CK), plasma lactate dehydrogenase
(LDH), perceived pain, knee range of movement, and thigh circum-
ference were measured. Muscle soreness protocol was performed
with a weight load equal to 80% of the maximum isotonic force in
4 sessions with 20 repetitions and 3-minute rest in between.
Main Outcome Measures: This study shows that 10-day
supplementation with 300 mg saffron significantly decreased the
CK and LDH concentrations (P , 0.0001). In the saffron group,
there was no decline in maximum isometric and isotonic forces after
eccentric exercise, but a significant decline in the isometric force was
observed in the control group (P , 0.0001). No pain was reported in
the saffron group, whereas the indomethacin group experienced pain
before 72 hours (P , 0.001).
Conclusions: Results obtained from the current novel research
indicate a strong preventive effect of 10-day supplementation with
saffron on the DOMS.
Submitted for publication August 7, 2013; accepted March 5, 2014.
From the Department of Physical Education and Sports Sciences, University of
Mohaghegh Ardabili, Ardabil, Iran.
Supported by the university postgraduate thesis grant M416.
A. Meamarbashi was an employee of University of Mohaghegh Ardabili at
the time of this study, and A. Rajabi was an MSc student during this study
in the University of Mohaghegh Ardabili.
Corresponding Author: Abbas Meamarbashi, PhD, Department of Physical
Education and Sports Sciences, University of Mohaghegh Ardabili,
Ardabil 56199-11367, Iran (a_meamarbashi@yahoo.com).
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Clin J Sport Med  Volume 25, Number 2, March 2015
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Clinical Relevance: The saffron can be used to prevent DOMS
and alleviate the DOMS symptoms.
Key Words: saffron, indomethacin, delayed-onset muscle soreness,
creatine kinase, lactate dehydrogenase, isometric and isotonic forces
(Clin J Sport Med 2015;25:105–112)
INTRODUCTION
Delayed-onset muscle soreness (DOMS) is associated
with pain and discomfort in the first few days after a strenuous
exercise session. Eccentric muscular contractions in downhill
running, hopping, plyometric exercise, squatting, and during
the lowering phase of lifting weights can produce DOMS.1
Athletes are concerned about muscular discomfort and pain
phenomena because it can limit their exercise and training
activity.2 The main symptoms in DOMS are muscular stiff-
ness, tenderness, and pain during active movements.3,4 There
are many symptoms related to the muscle inflammation and
damage such as muscle fibre swelling,5 elevated serum activ-
ities of muscle specific enzymes such as the creatine kinase
(CK) and lactate dehydrogenase (LDH),6,7 reduced muscle
strength,1 and knee joint range of movement (ROM).8
Eccentric exercise has series of effects in the cell
membrane, causing an inflammatory response that leads to
production of prostaglandin E2 and leukotrienes. The muscle
microscopic injury is instigated by a mechanical disruption to
sarcomeres,9 T-tubules, myofibrils, cytoskeletal protein, and
the sarcoplasmic reticulum,10–12 which leads to an inflamma-
tory response.13 After muscle injury, enzymatic reactions and
inflammatory mediators such as thromboxanes, prostaglandins,
and leukotrienes from the cyclooxygenase and lipoxygenase
pathways correspond to increases in vascular permeability
and pain perception by sensitizing the types III and IV afferent
nerve fibres to both chemical and mechanical stimuli.1
The magnitude of force loss after eccentric exercise has
been claimed to be the best indirect marker of muscle
soreness.14 Mechanism of maximal force reduction in DOMS
is thought to be secondary to sarcomere “popping” and
disorganization,15 as well as damage to components of the
excitation–contraction coupling process.16,17
Prevention and treatment strategies to alleviate the
symptoms and signs of DOMS are numerous and varied,
including pharmacological (eg, nonsteroidal anti-inflammatory
medications),18–20 exercise,19,21–24 stretching,19,21,22 whey pro-
tein,25 fish oil, isoflavones,26 caffeine,27 L-carnitine,28 herbs,29
antioxidant vitamins,30 cryotherapy,19,31–34 transcutaneous elec-
trical nerve stimulation,32 and ultrasound.24,35 However, these
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 Meamarbashi and Rajabi
methods had small or no effect on DOMS or were not consis-
tent in attenuating DOMS pain and other symptoms.1
Administration of aspirin and acetaminophen did not
reduce the DOMS and the CK response to eccentric exercise.36
Previous studies found that ibuprofen37 has no effect on muscle
soreness. In addition, several dietary supplements have been
tested in the treatment of DOMS, including whey protein,
vitamin C, protease enzymes, phosphatidylserine, chondroitin
sulfate, vitamin E, and fish oil, all with small effect or not
effective on DOMS.1,26,38–45 There is no clear consensus in
the existing literature on an intervention that can effectively
relieve pain after eccentric exercise. Conclusively, it is difficult
to recommend with confidence the use of these methods
for minimizing the signs and symptoms of muscle soreness.45
Some herbs and spices such as saffron are widely used in
the human diet. Saffron is a spice derived from the flower of
the Crocus sativus. Saffron has been traditionally used in
ancient medicine against various human diseases. Saffron
stigma contains more than 150 volatile and aroma-yielding
compounds. It also has many nonvolatile active components,46
many of which are carotenoids, including zeaxanthin, lyco-
pene, and various a- and b-carotenes.47 Recent experimental
findings indicate that saffron’s major compounds, crocin and
crocetin, which are derivatives of carotenoids, are powerful
antioxidants,48 with anti-inflammatory49 and antinocicep-
tive50,51 activities.
Nonsteroidal anti-inflammatory drugs (NSAIDs) such
as ibuprofen, indomethacin, and diclofenac are the most
commonly investigated treatment for DOMS. The risk of
overuse and its potential side effects such as stomach ulcers,
hepatic, and renal toxicity should be considered. NSAIDs
blocking cyclooxygenase enzyme thus inhibit the synthesis of
prostaglandins from arachidonic acid and therefore neutrophil
and macrophage functions are inhibiting.52 Previously, pre-
ventive effect of indomethacin (75 mg per day) for 5 days on
the cytokine responses to strenuous exercise in humans has
been studied.53 The results demonstrate that indomethacin
blunted serum intelukin-6 and augmented tumor necrosis
factor-alpha (TNF-a) and intelukin-10 during and after stren-
uous physical exercise. It is worthy to note that inflammation
itself is an essential natural process of muscle remodeling and
regeneration after muscle injury, and these medicines can
cause harm to the muscle.
Based on the anti-inflammatory and antinociceptive
effects of saffron and to compare any preventive effect of
saffron with indomethacin, therefore, this study aimed to
investigate the effects of 10-day supplementation with saffron
in DOMS in young male university students.
METHODS
The Physical Activity Readiness Questionnaire (PAR-Q)
was used for fitness and health screening of volunteers. Subjects
were excluded if they regularly participated in vigorous exercise
in the previous 3 months. Thirty-nine young healthy male
nonactive university students (age: 18.2 6 0.4 years) were
selected among 50 volunteers. This study used a 10-day, ran-
domized, double-blind, placebo-controlled, fixed-dose, parallel-
group, pretest–posttest design. The university ethics committee
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Clin J Sport Med  Volume 25, Number 2, March 2015
approved the study (M416). The purpose and procedures
of the study were fully explained, and they signed the con-
sent form.
The weight of the subjects, wearing minimal clothing
and standing height without shoes were measured with a Seca
digital scale (Seca model 707; Vogel & Halke GmbH & Co,
Hamburg, Germany) to the nearest 0.1 kg and 0.5 cm.
A dietary recommendation sheet was given to the
subjects and requested to refrain from any nutritional supple-
ments or medicines, including anti-inflammatory medicines, 1
week before and during the study. Crocus sativus L. stigma was
powdered, and soft capsules were filled with 300 mg saffron
powder. Contents of indomethacin capsules (25 mg) were
transferred to a similar 500 mg capacity capsule, and identical
placebo capsules containing 300 mg lactose were provided.
The saffron group received 1 capsule containing dried
saffron stigma powder (n = 12, 300 mg/d), indomethacin
group received 75 mg indomethacin (n = 12, 25 mg thrice
a day), and the control group (n = 15) received placebo cap-
sules containing 300 mg lactose powder. Participants were
asked to take the saffron and placebo capsules daily at 6 PM
with a cup of water and indomethacin every 8 hours imme-
diately after a meal.
A leg press apparatus equipped with 2 force isometric
sensors connected to a computerized dynamometer was used
to determine the maximum isometric force. Participants were
familiarized with the leg press machine and protocols before
testing.
First, all the participants underwent a 10-minute warm-
up. Then, each participant was tested for maximum isometric
force in a leg press machine. The maximum isometric force
was measured while the weight is locked and subject was
seated on the leg press bench, placed feet on the plantar plate
with knee angle at 908. Two computerized dynamometer sen-
sors measured the forces applied by the feet to the plantar
plates. Force data were recorded on a laptop computer and
software calculated the maximum isometric force during 3 to
5 seconds trial.
After 10-minute rest, maximum isotonic force was
measured when the weight bar was unlocked, and the subject
was seated on the bench with knee angle at 908 and pushed-up
the weights by feet. Verbal encouragement was given to
achieve a maximal effort isometric and isotonic strength tests.
After a 10-minute rest, participants were instructed to
perform 4 sets of eccentric exercise on a leg press machine.
The weight load was set to 80% maximum isotonic force in 4
sets. Each set consisted of 20 repetitions with 3-minute rest
between the sets. Participants were instructed to push the
weights up by extending legs and let it slowly swing back after
a short pause. Movements were harmonized by a metronome
sound. The participants performed the protocol with a maximal
effort until they were unable to continue a repetition with
proper technique. The force applied to the plantar plate was
recorded by a computerized dynamometer during the eccentric
exercise. All participants completed the protocol.
One day before the supplementation and 3 days after
eccentric exercise, the blood sample was taken after overnight
fasting from the control and experimental groups and before
other tests. Five milliliters of venous blood was sampled from
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 Clin J Sport Med  Volume 25, Number 2, March 2015
an antecubital vein and collected into a tube containing tri-
potassium EDTA. Samples were centrifuged at 3000 rates
per minute for 10 minutes to separate the plasma. Plasma
LDH and CK concentration were determined by an automatic
biochemistry analyser (Hitachi Model 902; Hitachi Ltd,
Tokyo, Japan) using commercial kits (Pars Azmoon, Tehran,
Iran).
Muscle swelling/inflammation was determined by mea-
suring the right thigh circumference. It was measured while
the subject was standing relaxed with the right leg slightly
apart to facilitate easy measurement. Mid-thigh circumference
was determined midway between the greater trochanter and
the lateral epicondyle of the femur using a plastic measure-
ment tape. The skin was marked with a semi-permanent
marker for consistency on subsequent days. The thigh
circumference of the right leg was measured 3 times to the
nearest 1 mm, and the averages were reported.
To measure the knee joint ROM, subjects laid prone on
an examination table with both knees fully extended. The
right knee joint angle was determined by using a goniometer
and universal landmarks (lateral epicondyle of the femur,
lateral malleous, and greater trochanter) to ensure alignment.
Landmarks were marked with a semi-permanent pen to ensure
consistency of subsequent measures. The axis of the goni-
ometer placed at the intersection of the right thigh and shank
at the knee joint. The goniometer stationary arm was along
the line from the knee joint to the greater trochanter. The
movable arm was along the lateral aspect of the fibula. Knee
flexion range was determined when the subject maximally
and voluntarily flexed his right knee.
A general rating of muscle pain was assessed using
a 0 to 6 point scale modified from that described by Talag.7,54
A score of “0” corresponded to a rating of “no pain,” whereas
a score of “6” corresponded to a rating of “unbearably pain-
ful.” Subjects were requested to rate the discomfort in only
the quadriceps and calf regions of the right leg, before, 24, 48,
and 72 hours after eccentric exercise.
The pretest and posttest data were obtained before the
supplementation and repeated 24, 48, and 72 hours after
eccentric exercise at 11 to 12 AM
Normal distribution of all the data was assessed using
the Shapiro–Wilk test. For each dependent variable, Lev-
enes test was used to determine homogeneity of variance,
with P , 0.05 indicating that the variance was not homo-
geneous. Changes in the measures in each group over time
were analyzed separately using a 2-way (group by time)
repeated-measures analysis of variance (ANOVA) with
Bonferroni pairwise comparisons. Measurements at before
the supplementation were used as baseline measurement
for the statistical analysis. The independent t test was used
for further comparison between 2 groups at each time
measurement.
Data were expressed as mean 6 SD. Values of P ,
0.05 were considered significant. The magnitude of the dif-
ference between pretest and posttest was assessed by using
Cohen’s d as the effect size. Cohen’s d of 0.20 is considered
a minor, 0.50 a medium, and 0.80 a major difference. Statis-
tical analysis was performed using SPSS 16.0 software (SPSS
Inc, Chicago, Illinois).
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Preventive Effects of Saffron and Indomethacin on DOMS
RESULTS
The Shapiro–Wilk test shows normality of all the data
distribution except for the pain perception in the pretest due to
pain-free status. Interpretation of data was performed using
a 2-way repeated-measure ANOVA with Bonferroni pairwise
comparison. Results obtained from the repeated-measure
ANOVA with Bonferroni pairwise comparison were used
for interpretation.
The maximum isotonic force showed equality in the
maximum force between all groups in the pretest session.
Conspicuously, in the control group, there was a significant
decline in the maximum isotonic force: after 24 hours
(15.3%), 48 hours (23.8%), and 72 hours (24.3%) after the
eccentric protocol. The maximum isotonic force between
saffron and control groups showed significant differences
after 24 (P , 0.05), 48 (P , 0.0001), and 72 hours (P ,
0.0001). The indomethacin and control groups comparison
had a significant difference after 48 (P , 0.05) and 72 hours
(P , 0.05). Comparison between saffron and indomethacin
showed the only significant difference after 24 hours (P ,
0.05). The effect size between saffron and control group after
24, 48, and 72 hours was 0.99, 1.7, and 2.1, respectively. The
effect size between indomethacin and control group after 24,
48, and 72 hours was 0.02, 0.19, and 0.23, respectively. The
saffron group had the highest effect size after 72 hours of
muscle soreness (Cohen’s d = 2.149) (Figure 1).
At baseline, there were no significant differences in
maximum isometric force between groups. The saffron group
exhibited significant increase in the isometric force after 24
(P , 0.05), 48 (P , 0.05), and 72 (P , 0.0001) hours (63.6%
increase) compared with baseline. The maximum isometric
force between saffron and control groups showed significant
differences after 24 (P , 0.05), 48 (P , 0.0001), and 72 hours
(P , 0.0001). Unlike saffron group, the control group showed
a significant decrease in the isometric and isotonic forces.
Comparison between saffron and indomethacin showed a sig-
nificant difference only after 72 hours (P , 0.05), whereas the
indomethacin and control groups comparison had a significant
difference after 48 and 72 hours (P , 0.05). The effect size
between saffron and control groups after 24, 48, and 72 hours
was 2.6, 2.9, and 2.6, respectively. The effect size between
indomethacin and control groups after 24, 48, and 72 hours
was 1.3, 0.28, and 0.29, respectively. The highest effect size
belonged to the saffron group compared with control after 72
hours of muscle soreness (Cohen’s d = 2.9) (Figure 2).
Plasma CK values were not significantly different
between the groups at baseline. However, Figure 3 demon-
strates that after 24 hours, CK concentration in the saffron and
indomethacin groups decreased as compared with the control
group. Plasma CK in the control group peaked at 48 hours
after exercise (125.8%). Plasma CK level in the saffron group
compared with baseline (100%) increased after 24 hours of
exercise but it was not significant (3.7%) after 24 hours of
exercise and then returned to a normal level. Plasma CK in
saffron and control groups was significantly different after 24,
48, and 72 hours (P , 0.0001). However, a difference
between indomethacin and control groups was significant
only after 48 and 72 hours (P , 0.0001). Saffron and
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 Meamarbashi and Rajabi Clin J Sport Med  Volume 25, Number 2, March 2015

FIGURE 1. Maximum isotonic force

changes between the saffron (♦),
indomethacin (n), and control (:)
groups (mean 6 SD). *Indicates
a significant difference (P , 0.05)
compared with the control group.
†Indicates a significant difference
(P , 0.0001) compared with the
control group.

indomethacin groups exhibited a significant difference only
after 24 hours (P , 0.005). The effect size between saffron
and control groups after 24, 48, and 72 hours was 2.6,
2.9, and 2.6, respectively. The effect size between indometh-
acin and control groups after 24, 48, and 72 hours was 1.3,
0.28, and 0.29, respectively. The maximum effect size has
seen between saffron and control groups after 24 hours
(Cohen’s d = 3.397).
There was no significant difference between groups
in the plasma LDH concentration at baseline. Plasma LDH
concentrations presented in Figure 4 indicate that the saf-
fron group exhibits a significant decrease after 24, 48, and
72 hours (P , 0.0001). The indomethacin group has a sig-
nificant difference with control group after 48 and 72 hours
(P , 0.0001). The saffron and indomethacin compa-
rison had a significant difference only after 24 hours

FIGURE 2. Comparison between

maximum isometric force in the
saffron (♦), indomethacin (n), and
control (:) groups (mean 6 SD).
*Indicates a significant difference
(P , 0.05) compared with the con-
trol group. †Indicates a significant
difference (P , 0.0001) compared
with the control group.

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 Clin J Sport Med  Volume 25, Number 2, March 2015 Preventive Effects of Saffron and Indomethacin on DOMS

FIGURE 3. Comparison between

plasma CK concentration in the saf-
fron (♦), indomethacin (n), and
control (:) groups (mean 6 SD).
*Indicates a significant difference
(P , 0.0001) compared with the
control group.

(P , 0.0001). The effect size between saffron and control
groups after 24, 48, and 72 hours was 2.6, 3.8, and 2.7,
respectively. The effect size between indomethacin and
control group after 24, 48, and 72 hours was 2.2, 0.61,
and 0.63, respectively. Highest level of effect size was
observed in the comparison between saffron and control
groups after 48 hours (Chohen’s d = 3.772).
Right leg pain perception had a significant difference
between saffron and control groups after 24, 48, and 72 hours
(P , 0.0001) (Figure 5). Pain perception in the saffron group
after 24 hours was 11.2 times lower than the control group,
and all the subjects in the saffron group experiences no pain
after 48 and 72 hours, whereas in the indomethacin group
pain alleviated after 72 hours.

FIGURE 4. Comparison between

plasma LDH concentration in the
saffron (♦), indomethacin (n), and
control (:) groups (mean 6 SD).
*Indicates a significant difference
(P , 0.0001) compared with the
control group.

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 Meamarbashi and Rajabi Clin J Sport Med  Volume 25, Number 2, March 2015

FIGURE 5. Comparison between

Talag scale perceived pain (left) in
the saffron (♦), indomethacin (n),
and control (:) groups (mean 6
SD). *Indicates a significant differ-
ence (P , 0.05) compared with the
control group. †Indicates a signifi-
cant difference (P , 0.0001) com-
pared with the control group.

The right thigh circumference did not show any signif-
icant differences at baseline. In the control group, the thigh
circumferences after 24, 48, and 72 hours of eccentric exercise
showed a significant increase as compared with the pretest.
Thigh circumference in the saffron group did not increase after
eccentric exercise. There was no significant difference between
the indomethacin and control groups (Table).
Control group exhibited a significant decrease in the
right knee joint ROM 24, 48, and 72 hours after exercise
compared with the pretest. The right knee joint ROM in
saffron and control groups had no significant differences after
24, 48, and 72 hours. However, comparison between
indomethacin and control groups showed a significant differ-
ence after 24 hours (P , 0.0001).
DISCUSSION
The present investigation demonstrated that saffron and
indomethacin markedly prevented the laboratory and clinical
indices of exercise-induced DOMS in the nonactive young
university students. The saffron was also more effective
compared with indomethacin in the prevention of DOMS.
The results of this study are encouraging because they serve
to highlight the potential importance of saffron as a natural
product in aiding the performance of athletes.
The best indicator of muscle damage is the force loss.14
In our experiment, the control group had a significant
decrease in the maximum isometric and isotonic forces
through the study. Noticeably, isometric force in the control
group significantly decreased (19.6%), whereas saffron group
experienced a significant increase after eccentric exercise
(63.6%). However, there were nonsignificant changes of max-
imum isometric force in the indomethacin group. This ten-
dency might provide a clue that saffron has double positive
effects on muscle, the preventive effect on DOMS, and mus-
cle force enhancement.
It has been proposed by Proske and Morgan55 that mus-
cle damage due to eccentric exercise leads to a release of Ca2+

TABLE. Participants’ Range of Motion of the Knee and Thigh Circumference Measurements Between the 3 Groups During the
Tests (Mean 6 SD)
Group Before 24 Hours 48 Hours 72 Hours
Thigh circumference Saffron 53.0 6 4.91 53.0 6 4.87 53.0 6 4.91 53.0 6 4.91
Indomethacin 54.1 6 4.70 54.7 6 4.72 54.0 6 4.70 54.0 6 4.70
Control 53.8 6 4.83 56.2 6 5.15* 57.1 6 4.81* 55.9 6 4.75†
Knee ROM Saffron 41.1 6 3.39 41.0 6 3.39 40.8 6 3.35 40.7 6 3.4
Indomethacin 35.2 6 2.5 36.1 6 2.51† 35.2 6 2.52 35.2 6 2.5
Control 36.7 6 2.0 39.0 6 2.07* 41.0 6 2.18* 39.5 6 2.1*
*Significant difference ,0.001.
†Significant difference ,0.0001.

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 Clin J Sport Med  Volume 25, Number 2, March 2015
in the sarcoplasm. The increased Ca2+ levels can trigger
proteolysis and assist in the break down of the damaged mus-
cle fibers. Moreover, damage due to increased myoplasmatic
Ca2+ levels can lead to inflammation, resulting in edema and
muscle swelling.55,56 The inflammation is assumed to sensitize
the nociceptors and leads to pain in the affected area.57,58 Our
previous report about the novel protective effect of saffron on
the erythrocyte osmotic hemolysis (76.0%)59 might support
this notion that saffron might protect the muscle fibers by
enhancing the muscle cell membrane integrity.
An increase in the maximum isometric force in the
saffron group is a novel finding of this study. In a very recent
and unpublished research, we found significant ergogenic
effect of saffron after 10-day saffron supplementation. We
postulated that this ergogenic effect of saffron might be
related to (1) enhance muscle metabolism, (2) increased
neuronal excitability, increased motor unit recruitment, (3)
combination of these mechanism with or without anti-
inflamatory and antinociceptive effects of saffron. Antioxi-
dant effects and the facilitation in tissue oxygenation
properties of saffron components (eg crocin, crocetin, and
safranal) were previously reported.60
Increases in plasma CK and LDH levels are established
indicators of inflammation and pathological consequences of
DOMS. Saffron showed better prevention of CK and LDH
elevation after eccentric exercise compared with indometha-
cin. Anti-inflammatory and antioxidant effects of saffron are
the most likely responsible. These effects may also be
responsible for the perceived pain tolerance in this study.
This study was limited to the inactive and healthy university
male students, and similar research on the athletes is
necessary to find out the effectiveness of saffron on the
athletes.
CONCLUSIONS
We conclude that 10-day supplementation with saffron
has potent preventive effects on DOMS as compared with
indomethacin and control groups. The ancient spice saffron
has shown eminent preventive effect on DOMS as compared
with indomethacin. Considering NSADs medicine side
effects, saffron with its health-promoting properties will be
preferred in the prevention of DOMS. Further research is
necessary to unravel the mechanisms involved and dose-
dependent effect of saffron on DOMS.
ACKNOWLEDGMENTS
The authors are most grateful to the participants in this
research study.
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