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Sport Sciences for HealthFounded by the Faculty of ExerciseScience - University of Milan, officialjournal of the Italian Society of Exerciseand Sport Sciences ISSN 1824-7490 Sport Sci HealthDOI 10.1007/s11332-014-0179-8

Does Ramadan fasting affect the diurnalvariations in metabolic responses and totalantioxidant capacity during exercise inyoung soccer players?

Omar Hammouda, Hamdi Chtourou,Asma Aloui, Mohamed Arbi Mejri,Henda Chahed, Abdelhedi Miled, KarimChamari, Anis Chaouachi, et al.

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ORIGINAL ARTICLE

Does Ramadan fasting affect the diurnal variations in metabolicresponses and total antioxidant capacity during exercise in youngsoccer players?

Omar Hammouda • Hamdi Chtourou • Asma Aloui •

Mohamed Arbi Mejri • Henda Chahed • Abdelhedi Miled •

Karim Chamari • Anis Chaouachi • Nizar Souissi

Received: 25 November 2013 / Accepted: 4 March 2014

� Springer-Verlag Italia 2014

Abstract The aim of this study was to investigate the

effects of Ramadan fasting and time-of-day on biochemical

responses to an intermittent exercise [Yo–Yo test level 1,

(YYIRT)]. Twenty male soccer players (17.52 ± 0.2 years,

177.4 ± 2.9 cm) completed the YYIRT at 0700 and

1700 hours on three occasions: 1 week before Ramadan

(BR), the second week of Ramadan (SWRR2), and the

fourth week of Ramadan (ERR4). The total distance cov-

ered during the YYIRT (TD) was recorded. Moreover,

blood samples were obtained before and after the YYIRT

for biochemical measurements. TD was higher BR than

during Ramadan in the evening (P \ 0.05), but not in the

morning. However, there was no significant difference

between BR and Ramadan in the morning. While post-

exercise values of blood lactate (Lac), glucose (GLC), and

markers of muscle injury were greater higher in the

evening, resting total antioxidant status (TAS) and uric acid

(UA) levels were higher in the morning as compared with

the evening BR. These diurnal variations were hidden

during Ramadan due to a significant decrease in Lac

(P \ 0.01), GLC (P \ 0.05) and cellular damage

(P \ 0.05) and an increase in TAS and UA (P \ 0.05)

values in the evening. No significant difference in bio-

chemical responses was observed in the morning during

SWRR2 and ERR4 as compared with BR. In summary, the

present study indicates that YYIRT performance was

affected by Ramadan fasting only in the evening in young

soccer players. The modified diurnal pattern of biochemical

responses could explain this performance decrement.

Keywords Fasting � Sport performance � Transaminases �Chronobiology � Soccer � Antioxidant

Introduction

Muslim athletes may have to train or compete during the holy

month of Ramadan. This religious tenet implies a total

abstinence from food and fluid intake from dawn to sunset.

Currently, there is little information regarding the effects of

Ramadan fasting (RF) on physiological responses in well

trained athletes [1–4]. Research suggested that Muslim ath-

letes are likely to experience a myriad of biochemical

adjustments that may lead to alterations in the hormonal,

immune, and antioxidant systems during Ramadan [3–6].

Despite this, there is no clear evidence of a major increase in

physiological stress or chronic systemic inflammation

throughout the fasting month [3, 4, 6]. Moreover, most of the

studies focused on resting levels of biochemical parameters,

and data exhibiting changes in biochemical responses to

exercise during Ramadan in trained subjects are lacking. In

O. Hammouda (&) � H. Chtourou � A. Aloui �M. A. Mejri � A. Chaouachi � N. Souissi

Research Laboratory ‘‘Sport Performance Optimization’’,

National Center of Medicine and Sciences in Sport (CNMSS),

Bp 326, Ave Med Ali Akid, El Menzah, 1004 Tunis, Tunisia

e-mail: omarham007@yahoo.fr

O. Hammouda � H. Chtourou

High Institute of Sport and Physical Education of Sfax,

Sfax University, Sfax, Tunisia

H. Chahed � A. Miled

Laboratoire de Biochimie, CHU Farhat Hached, Sousse, Tunisia

K. Chamari

Research and Education Center, Aspetar, Qatar Orthopaedic and

Sports Medicine Hospital, Doha, Qatar

N. Souissi

High Institute of Sport and Physical Education of Ksar-Saıd,

Manouba University, Manouba, Tunisia

123

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DOI 10.1007/s11332-014-0179-8

Author's personal copy

this context, Bouhlel et al. [1] observed that blood glucose

(GLC) values did not change after exercise; however, hae-

moglobin concentrations and haematocrit were significantly

higher at the end of Ramadan compared with before Rama-

dan. Recently, Aziz et al. [7] found that blood GLC levels

measured at rest were lower during Ramadan than before

Ramadan, while GLC and lactate (Lac) levels measured after

an intermittent exercise were not significantly different

between the two testing phases. Also, a decrease in urea

concentrations was observed after a submaximal exercise

performed during Ramadan, suggesting that there was no

increase in endogenous protein metabolism to compensate

the decreased protein intake [8, 9]. Furthermore, Chennaoui

et al. [5] showed that salivary cortisol concentrations mea-

sured after a maximal endurance exercise were higher in the

first week of Ramadan compared with before Ramadan, but

dropped by the end of the fasting month.

On the other hand, endurance training in a fasted state

has been shown to increase muscle oxidative capacity [10]

and enhance exercise-induced net intramyocellular lipid

degradation [11].

The discrepancies between studies regarding the meta-

bolic effects of RF are possibly linked to differences

between protocols, the time of blood sampling, and the

selection of the sampling day during Ramadan [12].

Likewise, it has been shown that RF affected the

circadian pattern of body temperature, cortisol, melato-

nin, and biochemical measures [e.g., glycemia, transam-

inases, creatine kinase (CPK)], but does not affect lipid,

carbohydrate, or protein metabolism, and mean daily

serum hormone levels [12–16]. Also, Waterhouse et al.

[17] reported a decrease in Lac concentrations during a

submaximal exercise performed in the evening without a

change in morning measures during Ramadan. The lower

evening Lac levels [17] could be explained by a higher

use of lipids as fuel at this time-of-day [16, 18, 19].

The determination of the circadian patterns of bio-

chemical parameters during Ramadan may provide an

explanation for the modified circadian pattern of sport

performance during this month.

In a previous research, we found that biochemical respon-

ses to the Yo–Yo intermittent recovery test level 1 (YYIRT)

were time-of-day dependant [20]. In the present study, we

aimed to examine the effect of RF on the diurnal variations in

some biochemical parameters in response to the YYIRT.

Methods

Participants

Twelve male professional soccer players (age

17.52 ± 0.2 years, height 177.4 ± 2.9 cm) volunteered to

participate in this study. They were affiliated with the same

club competing in the Tunisian first professional league [20]

and had a minimum of 5 years of training experience. The

participants trained at least four evening sessions per week

with an average of 2 h per session in addition to the weekend

match. During Ramadan, the training program consisted of

high intensity short-duration intermittent exercises. Goal-

keepers and players who experienced injuries were excluded

to have an homogeneous group in terms of physical char-

acteristics (e.g., strength, anaerobic power and capacity,

endurance). Participants were considered to be healthy on the

basis of a medical examination. Also, they were non-smok-

ers and did not consume caffeine or alcoholic beverages. To

be included in the study, each participant was requested to

keep standard times for eating (breakfast at 0700, lunch at

1200 hours, and dinner at 2000 hours) and sleeping habits

(sleeping between 2300 and 0700 ± 1 h) before Ramadan.

Moreover, participants were either of a ‘‘moderately morn-

ing type’’ (n = 4) or ‘‘neither type’’ (n = 8) based on their

responses to the Horne and Ostberg self-assessment ques-

tionnaire [21].

The experimental design was approved by the Clinical

Research Ethics Committee of the National Center of

Medicine and Sciences in Sport of Tunis and met the

ethical standards of the Declaration of Helsinki. The study

was conducted in Sfax (southeast of Tunisia, altitude

*7 m) in the summer of 2010 when the elapsed time from

dawn to sunset was from 0402 to 1910 hours at the

beginning and from 0431 to 1833 hours at the end of

Ramadan. During this period, the participants abstained

from food and drinks *16 h/day.

Experimental design

The experimental design consisted of three testing periods:

1 week before Ramadan (BR), the second week of Rama-

dan (R2), and the fourth week of Ramadan (R4). Body

mass (BM) and fat mass (FM) were measured at each

testing phase with an electronic balance (Tanita

TBF300WA, Tokyo, Japan). Furthermore, at each period,

the participants performed two test sessions at two times of

day (i.e., 0700 and 1700 hours), with a recovery period

C36 h in-between. Test sessions were completed in a

counterbalanced design. Each one commenced with oral

temperature measurement with a digital clinical ther-

mometer (Omron, Paris, France; accuracy ±0.05 �C).

Then, the soccer players performed an endurance specific

test (YYIRT). Heart rate was recorded during the YYIRT

using a Polar heart rate monitor (Polar Electro Oy, T61-

coded, Finland). Throughout the experimental period,

participants were requested to maintain their habitual

physical activities and to avoid strenuous activities *24 h

before each test session.

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Dietary records

A seven consecutive day dietary record was completed.

Dietary records were done in the same weeks as test ses-

sions (BR, R2, and R4). Participants received a detailed

verbal explanation and written instructions on data col-

lection procedures. They were asked to continue their usual

dietary habits during the period of diet recording and to be

as accurate as possible in recording the amount and types

of food and fluids consumed. A list of common household

measures (e.g., cups and tablespoons) and specific infor-

mation about the quantity of each measure (grams, etc.)

were given to each participant. Each individual’s diet was

calculated using the Bilnut 4 software package (SCDA

Nutrisoft, Cerelles, France) and the food composition

tables published by the Tunisian National Institute of Sta-

tistics in 1978.

Blood sampling and analysis

Fasting blood samples were collected from a forearm vein

after 5 min of rest in a seated position and 3 min after the

YYIRT. Samples were immediately placed into an ice bath

and then centrifuged for 10 min at 2,5009g and 4 �C.

Aliquots of the resulting plasma were stored at -80 �C

until analyzed. To eliminate inter-assay variance, all sam-

ples were analyzed in the same assay run. All assays were

performed in duplicate in the same laboratory with simul-

taneous use of a control serum from Randox Laboratories,

Ltd. (Crumlin, Co., Antrim, UK). Also, all reagents

employed in biochemical tests were obtained from Randox.

Aspartate aminotransferase (ASAT) and alanine ami-

notransferase (ALAT) activities were determined by an

enzymatic rate method. Blood GLC levels were measured

with the glucose oxidase method and Lac concentrations

were assessed by the lactate oxidase peroxidase method.

CPK activity was determined spectrophotometrically by

measuring NADPH formed by hexokinase and the D-glu-

cose-6-phosphate dehydrogenase coupled enzymatic sys-

tem. Lactate dehydrogenase (LDH) activity was

determined by measuring NADH consumption using

reagent kits. Uric acid (UA) concentrations were deter-

mined by an enzymatic method at 550 nm using a Randox

kit and protein concentrations (Pro) were determined by the

Biuret method.

Total antioxidant status (TAS) was measured using a kit

purchased from Randox. In this assay, metmyoglobin

reacts with H2O2 to form the radical species ferrylmyo-

globin. A chromogen [2,20-azinodi-(ethylbenzthiazoline

sulfonate); ABTS] is incubated with the ferrylmyoglobin to

produce the radical cation species ABTS?. This has a

relatively stable blue-green colour measured at 600 nm.

Antioxidants in the added sample cause suppression of this

colour production in proportion to their concentrations. The

detection limit for the TAS kit was 0.21 lmol/L.

The Yo–Yo intermittent recovery test level-1 (YYIRT)

As described previously [20], the test consisted of 20-m

shuttle runs performed at increasing velocities with 10 s of

active recovery between runs until exhaustion. The end of

the test was considered when the participant twice failed to

reach the front line in time (objective evaluation) or he felt

unable to complete another shuttle at the dictated speed

(subjective evaluation). The total distance (TD) covered

during the YYIRT (including the last incomplete shuttle)

was considered as the test score. All players were already

familiar with the test as it was part of their usual fitness

assessment program.

Statistical analyses

All statistical tests were processed using STATISTICA

Software (StatSoft, France). Mean and standard deviation

(SD) were calculated for each variable. The Shapiro–Wilk

W test of normality revealed that the data were normally

distributed. Once the assumption of normality was con-

firmed, parametric tests were performed. Oral temperature,

TD, and peak heart rate (HRpeak) data were analyzed using

a two-way analysis of variance (ANOVA) [3 (Rama-

dan) 9 2 (time-of-day)]. Also, a three-way ANOVA [3

(Ramadan) 9 2 (time-of-day) 9 2 (blood samples)] with

repeated measures was performed for the biochemical

measurements. When appropriate, significant differences

between means were tested using the Tukey post hoc test.

A probability level of 0.05 was selected as the criterion for

statistical significance.

Results

Body composition and dietary intake

BM and FM data are shown in Table 1.

Statistical analyses indicated that BM (P \ 0.001) and

FM (P \ 0.01) were lower during R4 than BR.

Compared with BR, the estimated daily energy intake was

substantially reduced during R4 (3,302 ± 709.8 vs.

2,693 ± 517.13 kcal/day respectively, P \ 0.01) (Table 1).

This change was associated with a decrease in fat intake

during R4 (P \ 0.05), while carbohydrate and protein

intakes did not differ between Ramadan and BR (Table 1).

Moreover, vitamin A, vitamin C, and vitamin E intakes were

unchanged throughout the study (Table 1).

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Temperature and YYIRT Performance

Oral temperature was higher in the evening compared with

the morning before and during Ramadan (P \ 0.001)

(Fig. 1). This increase was higher BR (?0.71 ± 0.32 �C)

than during R2 (?0.40 ± 0.28 �C) and R4 (?0.41 ±

0.32 �C). Oral temperature was not affected in the morn-

ing; however, there was a significant decrease in oral

temperature during R4 compared with BR in the evening

(P \ 0.05).

The TD covered during the YYIRT was higher in the

evening than the morning BR (P \ 0.05) (Table 2).

However, these diurnal variations were not found during

Ramadan. Moreover, the TD decreased significantly during

R2 (P \ 0.01) and R4 (P \ 0.001) compared with BR at

1700 hours. Also, HRpeak decreased during R2 and R4

compared with BR in the evening (P \ 0.05; Table 2).

Biochemical measurements

The biochemical parameters measured before and after the

YYIRT, in the morning and in the evening, during the three

testing phases, are displayed in Table 3.

BR, the values of the measured biochemical parameters

were higher after the exercise than resting levels in the

morning and in the evening (P \ 0.001). Moreover, except

for Lac, CPK (P \ 0.001), LDH (P \ 0.01), GLC

(P \ 0.001), ASAT (P \ 0.01), and ALAT (P \ 0.001)

values measured at rest were higher in the evening com-

pared with the morning. These diurnal variations were

maintained after the YYIRT. However, UA (P \ 0.01),

TAS (P \ 0.001), and Pro (P \ 0.001) values measured at

rest were higher in the morning than the evening. These

diurnal variations were not found after the exercise.

For R2, all parameters had higher values after the exercise

compared with resting levels in the morning and in the

evening (P \ 0.001). Similarly to BR, CPK (P \ 0.001),

LDH (P \ 0.05), GLC (P \ 0.001), ASAT (P \ 0.05), and

ALAT (P \ 0.05) values were higher in the evening than the

morning during R2. These diurnal variations were main-

tained after the exercise. Nevertheless, UA (P \ 0.05), TAS

(P \ 0.05), and Pro (P \ 0.05) values were lower in the

evening compared with the morning. Except for Pro, these

diurnal variations were not observed after the YYIRT.

During R4, the levels of all parameters were higher after

the YYIRT compared with resting values in the morning

and in the evening (P \ 0.001). However, no diurnal var-

iation was observed in resting values of the selected bio-

chemical parameters at this testing period. After the

Table 1 Body mass, fat mass, and dietary intakes recorded before Ramadan (BR), during the second week of Ramadan (R2), and during the

fourth week of Ramadan (R4)

BR R2 R4

Body mass (kg) 66.9 ± 13.4 65.02 ± 10.7** 64.13 ± 11.9***

Fat mass (kg) 16.4 ± 6.6 16.1 ± 9.5 15.3 ± 4.4**

Daily energy intake (kcal/day) 3,256 ± 660.1 2,875.7 ± 654* 2,705 ± 612.2**

Carbohydrates (g) 412.3 ± 135.1 401.2 ± 87.1 409.3 ± 86.4

Proteins (g) 95.9 ± 33 97.2 ± 40.2 96.6 ± 28.4

Fats (g) 109.4 ± 64.2 104.3 ± 27.1 100.9 ± 36.4*

Vitamin C (mg/day) 45.9 ± 29 44.8 ± 38 43.6 ± 49.8

Vitamin E (mg/day) 4.23 ± 1.9 5.1 ± 4.1 4.6 ± 2.4

Vitamin A (ER) 1,980.4 ± 2.6 1,815.5 ± 1.4 1,726.5 ± 2.3

Data are mean ± SD

* Significantly different compared with BR value at P \ 0.05

** Significantly different compared with BR value at P \ 0.01

*** Significantly different compared with BR value at P \ 0.001

Fig. 1 Oral temperature (�C) measured at 0700 and 1700 hours

before Ramadan (BR), during the second week of Ramadan (R2), and

during the fourth week of Ramadan (R4). Data are mean ± SD.

Asterisk significantly different from BR value at the same time-of-day

(P \ 0.05). Triple hash symbol significantly different from

0700 hours value for the same period (P \ 0.001)

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exercise, only LDH values were significantly higher in the

evening compared with the morning (P \ 0.01).

Regarding the Ramadan effect, CPK, ASAT, and ALAT

values measured before and after the YYIRT were lower

during R4 compared with BR in the evening (P \ 0.001).

Likewise, compared with BR, LDH values were lower

during R2 after the exercise (P \ 0.01) and during R4 both

before (P \ 0.001) and after (P \ 0.01) the exercise in the

evening hours. GLC values were lower during R2 and R4

compared with BR in the evening (P \ 0.001 for before

and after the exercise). Also, Lac values measured after the

YYIRT were lower during R2 and R4 than BR in the

evening (P \ 0.05). Compared with BR, UA values were

higher during R2 before the exercise (P \ 0.05) and during

R4 both before (P \ 0.05) and after (P \ 0.05) the exer-

cise in the evening. Furthermore, resting values of TAS

were higher during R4 than BR in the evening (P \ 0.01),

while post-exercise values were not significantly different

between the two testing phases at this time-of-day. Pro

values measured before and after the YYIRT were higher

during R2 and R4 compared with BR in the evening

(P \ 0.05).

Discussion

This study aimed to investigate the effects of RF on the

diurnal variations in metabolic responses to an intermittent

exercise in Tunisian soccer players. The present study’s

results confirmed that RF affects endurance performance

measured in the evening, but not in the morning. Moreover,

RF modified the diurnal pattern of resting and post-exercise

values of the studied biochemical parameters.

The present study’s findings indicated that BM and FM

were lower during R4 compared with BR. These results are

in agreement with previous research [2, 22], and could be

related to an increased use of lipids [1, 23] and a decrease

in fat intake during Ramadan. Nevertheless, the findings of

a recent well controlled study indicated that RF didn’t

ameliorate the oxidation during submaximal exercise [24].

As previously shown [25], this study demonstrated that

the TD covered during the YYIRT was unaffected in the

morning, but reduced in the evening during Ramadan. To

date, few studies have examined the effect of RF on the

diurnal variations in sport performance [25, 26]. The

modified diurnal pattern of body temperature during

Ramadan could explain the present results. Indeed, a cir-

cadian analysis, where values were obtained every 2 h,

showed a delay in the acrophase, a decrease in the ampli-

tude, and no variation in rectal temperature during Rama-

dan [27].

Concerning biochemical measures, and similarly to our

recent findings [28–30], the present results indicated that

resting and post-exercise levels of CPK, LDH, and ASAT

showed higher evening values BR. These findings could

explain, at least in part, the diurnal pattern of endurance

performance [20]. During R4, resting and post-exercise

levels of these parameters decreased in the evening. These

modifications suppressed the diurnal pattern of resting val-

ues and reduced the amplitude of post-exercise levels. Also,

Haouari-Oukerro et al. [13] observed a decrease in the

amplitude and a shift in the acrophase of hepatic and myo-

cellular enzymes (i.e., gamma-glutamyltransferase, alkaline

phosphatase, CPK, and glutamic transaminase) during

Ramadan. Moreover, recent findings demonstrated that RF

did not adversely affect cellular damage [31]. In this context,

it has been shown that oxidative stress [31] and inflammatory

status (i.e., IL-6, C-reactive protein) [32] were reduced

during Ramadan in healthy participants. The decrease in

muscle damage observed during Ramadan could be, in part,

explained by the caloric restriction observed in the present

study (reduction in daily energy intake).

Consistent with our recent findings [20, 28–30], resting

and post-exercise levels of GLC and Lac were higher at

1700 hours as compared with 0700 hours BR. The morning

to evening difference in Lac response to exercise might be

Table 2 Total distance covered during the YYIRT (TD) and peak heart rate during the YYIRT (HRpeak) measured at the two times of day before

Ramadan (BR), during the second week of Ramadan (R2), and during the fourth week of Ramadan (R4)

BR R2 R4

0700 hours 1700 hours 0700 hours 1700 hours 0700 hours 1700 hours

TD (m) 1,746.53 ± 527.36# 1,947.64 ± 458 1,717 ± 554.35 1,798.47 ± 410.13** 1,722.43 ± 497.3 1,658.17 ± 522.13***

HRpeak

(beats/min)

191.4 ± 5.3 193 ± 5.9 189.4 ± 5.5 189 ± 3.1* 187.7 ± 3.5 187.6 ± 2.8*

Data are mean ± SD

* Significantly different from BR value at the same time-of-day (P \ 0.05)

** Significantly different from BR value at the same-time-of day (P \ 0.01)

*** Significantly different from BR value at the same time-of-day (P \ 0.001)# Significantly different from 1700 hours value for the same period (P \ 0.05)

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explained by the increase in catecholamine levels [33] and/

or the circadian variation in core temperature [34]. How-

ever, there was no diurnal variation in GLC and Lac levels

measured at rest or after the YYIRT during R4 because of

the decrease in evening values. Similarly, previous studies

showed that resting GLC levels increased at 02:30 and

0800 hours and decreased at 17:30 hours during Ramadan

[14, 35]. Concerning the exercise effect, recent research

found that Lac levels decreased after a submaximal exer-

cise performed during R4 in the evening [17]. However, a

more recent research concluded that while Lac values

remained unchanged, GLC levels were reduced during

evening submaximal exercise [24]. These results could be

explained by the modified circadian distribution of GLC

levels during Ramadan [14]. Moreover, the lower Lac

values reported during R4 in the evening could be attrib-

uted to a greater use of lipids as fuel at this time [16, 18,

19]. This fact was not confirmed in the study of Aziz et al.

[24] which concluded that Ramadan fasting didn’t ame-

liorate fat burn during evening exercise despite a decrease

in GLC levels. Furthermore, Hargreaves [36] showed that

low endogenous concentrations of muscle glycogen and

hypo-hydration are possible factors leading to lower blood

GLC and blood Lac concentrations during Ramadan.

However, a recent study indicated that only resting GLC

levels decreased, while post-exercise values of Lac and

GLC did not significantly change after an endurance

exercise performed during Ramadan [7, 37]. Despite a

decrease in carbohydrate intake during Ramadan, morning

GLC levels did not change, which may be due to an

upregulation of gluconeogenesis.

Regarding protein metabolism, and similarly to previous

research [35], Pro values measured before and after the

YYIRT were higher during R2 and R4 compared with BR in

Table 3 Biochemical

parameters measured before and

after the YYIRT, at the two

times of day, before Ramadan

(BR), during the second week of

Ramadan (R2), and during the

fourth week of Ramadan (R4)

Data are mean ± SD$ Significant difference

between the morning and the

evening

* Significant difference

between before and after the

exercise# Significant difference

compared with before Ramadan

Before exercise After exercise

0700 hours 1700 hours 0700 hours 1700 hours

BR

CPK (IU/L) 156.09 ± 43.97 179.91 ± 57.67$ 177 ± 51.62* 204.55 ± 67.37$,*

LDH (IU/L) 350.65 ± 40.74 410.83 ± 35.78$ 420.27 ± 40.05* 557.45 ± 111.51$,*

GLC (mmol/L) 4.36 ± 0.09 4.7 ± 0.4$ 5.12 ± 0.08* 5.6 ± 0.29$,*

Lac (mmol/L) 1 ± 0.23 1.09 ± 0.16 11.14 ± 0.59* 12.03 ± 0.37$,*

UA (lmoL/L) 265.88 ± 38.64$ 243.56 ± 42.72 307.39 ± 46.48* 295.93 ± 38.35*

TAS (lmol/L) 1.23 ± 0.2$ 1.09 ± 0.15 1.36 ± 0.15* 1.3 ± 0.14*

Pro (g/L) 77.09 ± 8.29$ 67.09 ± 4.13 81 ± 9.97$,* 72.82 ± 6.71*

ASAT (IU/L) 24.8 ± 4.26 31 ± 4.69$ 31.4 ± 5.91* 37.2 ± 6.68$,*

ALAT (IU/L) 19.78 ± 2.11 22.78 ± 4.27$ 26.11 ± 4.34* 29.78 ± 3.56$,*

R2

CPK (IU/L) 145.27 ± 29.42 168.09 ± 46.74$ 164.73 ± 31.42* 194.27 ± 49.16$,*

LDH (IU/L) 359.56 ± 69.37 401.55 ± 47.66$ 456.24 ± 77* 504.95 ± 44.09$,*,#

GLC (mmol/L) 4.26 ± 0.22 4.48 ± 0.3$,# 5.06 ± 0.57* 5.56 ± 0.67$,*,#

Lac (mmol/L) 0.96 ± 0.13 0.98 ± 0.12 10.16 ± 0.6* 11.01 ± 0.4$,*,#

UA (lmoL/L) 276.95 ± 22.36$ 262.02 ± 37.5# 308.12 ± 35.05* 297.73 ± 39.52*

TAS (lmoL/L) 1.27 ± 0.08$ 1.18 ± 0.09 1.38 ± 0.07* 1.32 ± 0.07*

Pro (g/L) 76.18 ± 10.49$ 72 ± 4.94# 80.09 ± 9.62$,* 75.45 ± 5.91#

ASAT (IU/L) 25.9 ± 2.42 30.1 ± 3.57$ 35.7 ± 3.89* 40.4 ± 2.84$,*

ALAT (IU/L) 18.56 ± 3.24 20.89 ± 3.69$ 24.56 ± 4.95* 26.67 ± 5.15$,*

R4

CPK (IU/L) 142.91 ± 25.07 150.36 ± 28.27# 161.45 ± 40.93* 182.36 ± 52.09$,*,#

LDH (IU/L) 350.47 ± 27.78 373.13 ± 30.83# 436.71 ± 43.44* 494.06 ± 37.15$,*,#

GLC (mmol/L) 4.38 ± 0.33 4.48 ± 0.25# 5.03 ± 0.39* 5.12 ± 0.39*,#

Lac (mmol/L) 0.96 ± 0.2 1.02 ± 0.09 10.53 ± 0.55* 10.63 ± 0.28*,#

UA (lmoL/L) 281.7 ± 17.16 270.2 ± 16.66# 313.86 ± 23.15* 311.83 ± 28.06*,#

TAS (lmoL/L) 1.24 ± 0.07 1.2 ± 0.04# 1.38 ± 0.12* 1.35 ± 0.1*

Pro (g/L) 77 ± 9.6 75.09 ± 3.59# 80.64 ± 7.78* 77.36 ± 7.19#

ASAT (IU/L) 25.9 ± 3.81 26.4 ± 3.69# 36 ± 3.74* 33.8 ± 3.19*,#

ALAT (IU/L) 17.89 ± 2.8 18.89 ± 2.42# 25.89 ± 2.42* 26.67 ± 2.18*,#

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the evening. Moreover, consistent with our recent findings

[20, 28–30], the present results showed that resting and

post-exercise levels of UA, the end product of purine

breakdown, displayed higher morning values BR. More-

over, resting and post-exercise levels of UA increased sig-

nificantly during Ramadan. The results of the well-

controlled studies in athletes suggested that there is an

increased rate of protein breakdown and fat oxidation dur-

ing Ramadan [1, 2, 4]. Besides, the increased UA concen-

trations observed during Ramadan could be linked to

dehydration [1, 2, 38] and an excessive breakdown of RNA

tissue [39]. Furthermore, in the present study, the morning

to evening difference in UA concentrations was reduced

during R2 and was not observed during R4 by an increase in

evening values. This may indicate a better antioxidant status

at this time-of-day during R4. In addition, resting TAS

values increased during R4 in the evening, while post-

exercise levels were unchanged throughout the study. The

increase in TAS could be due to the caloric restriction

induced by RF. To date, few studies have examined the

effects of RF on oxidative stress biomarkers. Ibrahim et al.

[31] observed a reduction in malondialdehyde, without a

significant change in carbonylated proteins, glutathione, as

well as glutathione peroxidase and catalase activities.

However, a more recent study showed that 15-F2t-iso-

prostane, an urinary marker of oxidative stress, increased at

the end of Ramadan proportionally to BM and FM [40].

The timing of food availability and intake exerted a

powerful effect on the temporal characteristics of biochem-

ical rhythmic phenomena [41]. In this context, chronobio-

logical studies have shown that RF affects the circadian

distribution of body temperature, cortisol, melatonin, and

glycemia; but does not affect the metabolism of lipids, car-

bohydrates, or proteins, or mean daily serum hormone levels

[15, 16]. In this context, Cerizza et al. [42] concluded that a

proper lifestyle, in terms of dietary and physical habits and in

terms of psychological motivations for eating, avoids young

people becoming overweight and obese.

One limitation of this study was the relatively small

sample size and the lack of an untrained control group.

Moreover, blood sampling was done only before and after

the YYIRT. Further studies should investigate a delayed

blood sample to check peak values.

Conclusion

In summary, the present study’s results indicate that RF

modifies the diurnal pattern of resting and post-exercise

levels of the studied biochemical parameters. The con-

comitant decrease in the levels of biochemical markers of

muscle damage, Lac, and GLC at 1700 hours could explain

the modified diurnal pattern of endurance performance in

soccer players. It seems that the caloric restriction that

characterizes RF could influence the biochemical responses

and their diurnal pattern.

Acknowledgments This study was supported by the Ministry of

Higher Education and Scientific Research, Tunisia. We are grateful to

all the players who have so willingly participated in the study.

Conflict of interest The authors O. Hammouda, H. Chtourou, A.

Aloui, M.A. Mejri, H. Chahed, A. Miled, K. Chamari, A. Chaouachi

and N. Souissi declare that they have no conflicts of interest.

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