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Time-of-day effects on biochemical responses to
soccer-specific endurance in elite Tunisian football
players
Omar Hammouda a , Hamdi Chtourou a , Anis Chaouachi a , Henda Chahed b , Hlima
Bellimem b , Karim Chamari a c & Nizar Souissi a c
a Research Laboratory ‘Sport Performance Optimisation’, National Centre of Medicine and
Science in Sport, Sfax, Tunisia
b Laboratory of Biochemistry, CHU Farhat Hached, Sousse, Tunisia
c High Institute of Sport and Physical Education, Ksar-Saïd, Manouba University, Tunis,
Tunisia
Version of record first published: 14 Jan 2013.
To cite this article: Omar Hammouda , Hamdi Chtourou , Anis Chaouachi , Henda Chahed , Hlima Bellimem , Karim Chamari
& Nizar Souissi (2013): Time-of-day effects on biochemical responses to soccer-specific endurance in elite Tunisian football
players, Journal of Sports Sciences, DOI:10.1080/02640414.2012.757345
To link to this article: http://dx.doi.org/10.1080/02640414.2012.757345
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Time-of-day effects on biochemical responses to soccer-specific
endurance in elite Tunisian football players
OMAR HAMMOUDA
1
, HAMDI CHTOUROU
1
, ANIS CHAOUACHI
1
, HENDA CHAHED
2
,
HLIMA BELLIMEM
2
, KARIM CHAMARI
1,3
, & NIZAR SOUISSI
1,3
1
Research Laboratory ‘Sport Performance Optimisation’, National Centre of Medicine and Science in Sport, Sfax, Tunisia,
2
Laboratory of Biochemistry, CHU Farhat Hached, Sousse, Tunisia, and
3
High Institute of Sport and Physical Education,
Ksar-Saı¨d, Manouba University, Tunis, Tunisia
(Accepted 6 December 2012)
Abstract
This study aimed to investigate footballers’ diurnal variation of performance during the Yo-Yo intermittent recovery test and
the associated biochemical responses. Fifteen male footballers (17.3 +0.3 years, 69.1 +4.2 kg, 179.7 +3.6 cm) performed
two randomised Yo-Yo tests at 07:00 h and 17:00 h. Blood samples were collected before and 3 min after each test for the
assessment of metabolic responses. Resting oral temperature and rating of perceived exertion (RPE) after and peak heart rate
during the Yo-Yo test were recorded at both times-of-day. Core temperature and performances during the Yo-Yo test
increased from the morning to the evening (P50.0005 and P¼0.01, respectively) without significant time-of-day effects on
peak heart rate and RPE. Moreover, pre- and post-Yo-Yo test biochemical parameters (high-density lipoprotein,
triglycerides, glucose, creatine-kinase) were higher at 17:00 h than 07:00 h (160.45 +18.68 vs. 173.73 +14.48 before and
191.18 +21.13 vs. 219.27 +27.74 IU L
71
after the Yo-Yo test at 07:00 h and 17:00 h, P¼0.032 and P50.0005,
respectively for creatine-kinase). Only post-exercise lactate levels were higher in the evening (9.82 +0.65 vs.
10.86 +0.33 mmol L
71
,P50.0005) with all biochemical variables being increased after the exercise (P50.0005).
These findings suggest a possible link between the diurnal fluctuation of metabolic responses and the related pattern of
specific-endurance performances in footballers. Therefore, the higher biochemical responses observed in the evening could
explain, partially, the greater performance and metabolic solicitation at this time-of-day.
Keywords: lipid profile, intermittent endurance performance, circadian rhythm
Introduction
Recently, diurnal fluctuations in response to short-
term exercise involving anaerobic metabolism have
been confirmed (Chtourou, Chaouachi, Hammou-
da, Chamari, & Souissi, 2012; Chtourou, Hammou-
da, Chaouachi, Chamari, & Souissi, 2012;
Chtourou, Hammouda, Souissi, et al., 2012;
Chtourou, Zarrouk, et al., 2011; Hammouda et al.,
2011; Souissi, Gauthier, Sesboue, & Davenne,
2004). However, researches about time-of-day effect
on aerobic performance are yet inconclusive
(Chtourou, Chaouachi, Driss, et al., 2012; Chtour-
ou, Driss, et al., 2012; Chtourou & Souissi, 2012). A
time-of-day effect on oxygen uptake has been
identified at rest, during submaximal exercise, and
at the lactate threshold, but not on maximal oxygen
uptake (Brisswalter, Bieuzen, Giacomoni, Tricot, &
Falgairette, 2007; Forsyth & Reilly, 2004; Reilly,
Atkinson, & Waterhouse, 1997). However, other
findings showed no time-of-day effects on oxygen
uptake kinetics at the onset of moderate or high
intensity exercise (Carter, Jones, Maxwell, & Doust,
2002).
Furthermore, professional football matches are
played at various times-of-day, ranging from morn-
ing kick-offs to night-time competitions under flood-
lights. In this context, it has been recently found that
football players perform at an optimum between
16:00 h and 20:00 h when not only football-specific
skills (e.g., juggling performance, wall-volley test,
etc.) but also measures of physical performance (e.g.,
vertical jump, grip strength, etc.) are at their peak
(Reilly et al., 2007). Moreover, it has been shown
that mental performance is greater in the evening
hours (i.e., alertness and reaction time was highest
Correspondence: Omar Hammouda, Research Laboratory ‘Sport Performance Optimisation’, National Center of Medicine and Science in Sport, Sfax, Tunisia.
E-mail: omarham007@yahoo.fr
Journal of Sports Sciences, 2013
http://dx.doi.org/10.1080/02640414.2012.757345
Ó2013 Taylor & Francis
Downloaded by [Universite Laval] at 09:37 14 January 2013
and fatigue score was lowest at this time-of-day)
(Reilly et al., 2007). Particularly, the Yo-Yo inter-
mittent recovery test was specifically designed to
evaluate the ability to perform high-intensity inter-
mittent exercise in football players (Krustrup et al.,
2003). The test is extensively utilised by scientists
and coaches when monitoring cardio-respiratory
fitness of football players since it correlates with
match physical performance (Bangsbo, Iaia, &
Krustrup, 2008; Krustrup et al., 2003). Indeed, the
physiological measurements performed during the
Yo-Yo test showed that aerobic energy turnover
reached maximal values and that the anaerobic
energy system was highly taxed toward the end of
the test, making it a suitable test for football players
(Bangsbo et al., 2008; Krustrup et al., 2003). To the
authors’ knowledge, there seems to be only one study
about the diurnal variation of performance during
the Yo-Yo test in which our group showed that the
total distance covered during the test improved
significantly from the morning to the evening
(Hammouda, Chtourou, Farjallah, Davenne, &
Souissi, 2012). On the other hand, diurnal variation
of blood lactate responses that has been identified
during mild (Waterhouse, Alabed, Edwards, &
Reilly, 2009), maximal aerobic (Forsyth & Reilly,
2004, 2005) and anaerobic (Hammouda et al., 2011;
Hammouda, Chtourou, Chahed, et al., 2012)
exercises, would be indicative of raised anaerobic
metabolic activity in the evening (Thomas & Reilly,
1975). Indeed, as rhythms of physical performances
(Souissi et al., 2004) and biochemical responses
(Hammouda, Chahed, et al., 2012) are correlated
to the rhythmicity of oral temperature, the investiga-
tion of the time-of-day effects on biochemical
responses in the Yo-Yo test could explain the diurnal
pattern of the soccer-specific endurance test. In
this context, our group has recently identified the
diurnal variation of various biochemical markers at
rest (Hammouda, Chahed, et al., 2012) as well as
during repeated sprints (Hammouda et al., 2011)
and the Wingate test (Hammouda, Chtourou,
Chahed, et al., 2012) in trained football
players. However, to the best of our knowledge, the
diurnal variation of metabolic performance during a
soccer-specific endurance exercise has not been
studied yet. Therefore, the aim of this work was to
investigate the time-of-day effect on biochemical
responses to the Yo-Yo test in Tunisian football
players.
Methods
Participants
Fifteen male football players (17.3 +0.3 years,
69.1 +4.2 kg, 179.7 +3.6 cm; mean +s) volunteered
to participate in this study. The participants were
recruited on the basis of: (i) they trained, in the
evening, at least 4 days per week for an average of
2 h daily in addition to the weekend match and (ii)
they had a minimum 5 years of training experience
and were members of the same youth team
competing in the first division of the Tunisian
football league. We interviewed all players and
coaches in order to provide information concerning
the number of years of football practice and hours of
regular training per week. Moreover, goalkeepers
and players who experienced injuries were excluded
from the analysis. Approval for the study was
obtained from the club. Furthermore, after receiving
a thorough explanation of the possible risks and
discomforts associated with the experimental pro-
cedures, the participants provided written informed
consent to take part to the experiment. Answers to
the Horne and O
¨stberg (1976) questionnaire cate-
gorised participants as either ‘‘moderately morning’’
(n¼4) or ‘‘intermediate’’ (n¼11) chronotypes. No
participant reported tobacco use within the 6
months prior to the study, and none were taking
antioxidant compounds, including vitamins and
medications (e.g., anti-inflammatory agents). The
experimental design of the present study was
approved by the Clinical Research Ethics Commit-
tee of the National Centre of Medicine and Science
of Sports (CNMSS), Tunisia and met the ethical
standards of the Declaration of Helsinki.
Experimental design
Participants were familiar with the Yo-Yo test
protocol as it was regularly scheduled in their usual
test battery. They performed two test sessions, in a
randomised order, over 2 days with only one test
session per day, allowing a recovery period 36 h in-
between. One session was conducted in the morning
(07:00–08:30 h) and the other in the evening (17:00–
18:30 h). Since the experiment was carried out in the
summer, the two sessions coincide with the light
phase of the day. Moreover, these time points were
chosen because they correspond to the peak of
football-specific skills and measures of physical
performance (Reilly et al., 2007). In addition, these
time points span the portion of the day when people
typically participate in physical activity and training.
Upon arrival for their first test session, participants’
body mass (Tanita, Tokyo, Japan) and height
(Stadiometer, QuickMedical) were recorded. Fasting
blood samples were collected before and 3 min after
each test for the assessment of metabolic responses.
Moreover, oral temperature was measured with a
calibrated and valid digital clinical thermometer
(Omron, Paris, France; accuracy: 0.058C) inserted
sublingually for at least 3 min with the participants in
2O. Hammouda et al.
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a seated resting position for at least 15 min before
measurement. Although rectal temperature is usually
preferred as a marker of the body clock, this method
presented problems of social acceptability involved in
the present study. Therefore, in the present study,
only the oral temperature method was accepted by
the participants. Participants were asked to keep, as
closely as possible, their usual sleeping habits, with a
minimum of 7 h of sleep taken on the night
preceding each test session. Before the morning
test session, they were instructed to wake up at
06:00 h. They were fasting and allowed to drink only
one glass of water to avoid the effects of postprandial
thermogenesis (Bougard, Bessot, Moussay, Sesbou¨e´,
& Gauthier, 2009). Moreover, they were requested
to ingest a standardised meal at least 4 h before the
evening test session, as recommended by Bougard
et al. (2009) and not eating anything else until the
end of the testing session. Throughout the experi-
mental period, participants were requested to main-
tain their habitual physical activity and to avoid
strenuous activity during the 24 h before the test
sessions.
The Yo-Yo intermittent recovery test
As previously described by Chtourou, Hammouda,
et al. (2011), the Yo-Yo test was performed
according to the procedures suggested by Krustrup
et al. (2003). The reliability of the Yo-Yo test level-1
was established in a previous study (Castagna,
Impellizzeri, Cecchini, Rampinini, & Barbero,
2009). Indeed, Castagna et al. (2009) reported a
significant correlation (r¼0.65) between the total
distance covered during the Yo-Yo test and the total
distance covered during the soccer match. The test
consisted of 20-m shuttle runs performed at increas-
ing velocities with 10 s of active recovery between
runs until exhaustion. Audio cues of the Yo-Yo test
were recorded on a CD (www.teknosport.com,
Ancona, Italy) and broadcasted using a calibrated
portable CD player (Philips, Az1030 CD player,
Eindhoven, Holland). The end of the test was
considered when the participant twice failed to reach
the front line in time (i.e., objective evaluation) or he
felt unable to complete another shuttle at the
dictated speed (i.e., subjective evaluation). The total
distance covered during the Yo-Yo test (including
the last incomplete shuttle) was considered as the
test score. Before the test, all participants carried out
a warm-up period consisting of the first four running
bouts in the test. The total duration of the test was 6–
20 min. Heart rate was recorded during the Yo-Yo
test, using a Polar heart rate monitor (Polar Electro
Oy, T61-coded, Hungary, with values recorded each
5 s) and only peak heart rate was presented in the
data.
Rating of perceived exertion scale (RPE; Borg, 1982)
The RPE scale allows participants to give a subjective
exertion rating for the physical task (Chtourou,
Jarraya, Aloui, Hammouda, & Souissi, 2012). The
scale presents a 15-point scale ranging from 6 (very
very light) to 20 (very very hard). The RPE scale is a
reliable indicator of physical discomfort, has sound
psychometric properties, and is strongly correlated
with several other physiological measures of exertion
(Borg, 1982).
Dietary records
To assess the adequacy of nutrient intake, a 7-day
consecutive dietary record was completed. All
players received a detailed verbal explanation and
written instructions on data collection procedures.
Participants were asked to continue with their usual
dietary habits during the period of diet recording,
and to be as accurate as possible in recording the
amount and type of food and fluid consumed. A list
of common household measures, such as cups and
tablespoons, and specific information about the
quantity in each measurement (grams, etc.) were
given to each participant. Each participant’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 Statistics in 1978. Estimated
nutrient intakes were referred to reference dietary
intakes for physically active people (Aounallah-Skhiri
et al., 2011; Otten, Hellwig, & Meyers, 2006). The
data about the daily nutriment intake are presented
in Table II and showed that total calorie, macro-
nutrient, and micronutrient intakes are situated in
the interval of the reference dietary intakes for
healthy Tunisian adults.
Blood sample variable analysis
Glucose levels were measured with the glucose
oxidase method, and lactate concentrations were
measured by the lactate oxidase peroxidase method.
The coefficients of variation for these parameters
were 58%. Creatine-kinase activity was determined
spectrophotometrically by measuring nicotinamide
adenine dinucleotide phosphate formed by hexoki-
nase and the Dglucose-6-phosphate dehydrogenase
coupled enzymatic system. The intra-assay coeffi-
cient of variation for the creatine-kinase kit was
1.85%. Lactate dehydrogenase activity was
determined by measuring nicotinamide adenine
dinucleotide consumption using the reagent kits.
The intra-assay coefficient of variation for the lactate
dehydrogenase kit was 2.61%. Uric acid was
determined by an enzymatic method at 550 nm
using a Randox kit (Randox, Antrim, UK). The
Diurnal variation of biochemical responses in soccer players 3
Downloaded by [Universite Laval] at 09:37 14 January 2013
coefficient of variation for uric acid was 51.9%.
Moreover, total cholesterol, triglyceride, and high-
density lipoprotein cholesterol were estimated by
standard enzymatic analysis using reagents, stan-
dards, and controls from Randox Laboratories Ltd.
(Antrim, UK). The coefficients of variation for these
parameters were 57%. All the above measures were
carried out as adapted for the autoanalyser by
Synchron CX systems (Beckman Instruments, Dan-
ville, California, USA). All reagents employed in the
biochemical tests were obtained from Randox
Laboratories. Venous samples were corrected for
plasma volume changes, using the equations of Dill
and Costill (1974). Haematocrit was measured on
the same day as the experiment by microcentrifuga-
tion. Creatine-kinase and lactate dehydrogenase
were assessed because they reflect muscle damage.
Moreover, a positive correlation between markers of
muscle damage and free-radical production has been
previously shown which confirms the hypothesis that
free radicals produced during exercise alter muscle
cell membrane permeability (McBride & Kraemer,
1999). Moreover, uric acid was measured to assess
the purine cycle activation and to estimate the
antioxidant activity (Antoncic-Svetina et al., 2010).
In addition, glucose and lactate levels were measured
to assess muscle fatigue and glycolysis. Finally, lipid
profile (i.e., high-density lipoprotein, total cholester-
ol, and triglyceride) was estimated to quantify the
average of lipid mobilisation after exercise.
Statistical analyses
All statistical tests were processed using STATIS-
TICA Software (StatSoft, France). All values are
expressed as mean +s. The Shapiro-Wilk W-test of
normality revealed that the data were normally
distributed. Once the assumption of normality was
confirmed, parametric tests were performed. Biolo-
gical parameter data were analysed using a two-way
analysis of variance (ANOVA) (2 [Time-of-day] 62
[before/after Yo-Yo test]) with repeated measures.
When appropriate, significant differences between
means were assessed using the Fisher’s post-hoc tests.
The mean confidence interval was determined at
95%. Peak heart rate and total distance during the
Yo-Yo test, oral temperature, and RPE at the
different times-of-day were compared using paired
Student t-tests. The Pearson product-moment cor-
relation coefficients were used to determine whether
there was a significant relationship between lactate
and total distance covered during the Yo-Yo test. A
probability level of 0.05 was selected as the criterion
for statistical significance. Exact Pvalues were given;
however, results given as ‘‘P50.000’’ in the
statistics output were reported as ‘‘P50.0005’’.
Results
Core temperature, RPE, and Yo-Yo test performance
Table I presents the results of core temperature, the
total distance and the peak heart rate during the Yo-
Yo test, and the RPE scores after the Yo-Yo test
calculated in the morning and the evening. The
results showed that core temperature and total
distance during the Yo-Yo test increased between
the morning and the evening (P50.0005 and
P¼0.01, respectively). Concerning the peak heart
rate and RPE during the Yo-Yo test, no significant
time-of-day effect has been observed.
Dietary records
The mean daily calories, protein, carbohydrate, fat,
cholesterol, vitamin E, and vitamin A intakes were in
the normal ranges. However, the percentage of
protein intake is relatively small. These data are
presented in Table II.
Selected biochemical parameters
Table III shows the statistical results from analysis of
variance and the mean values for the selected
biochemical parameters at the two times-of-day,
before and after the Yo-Yo test.
As this table indicates, there were significant main
effects for time-of-day and before/after Yo-Yo test for
all biochemical parameters. Likewise, for all para-
meters, the interaction time-of-day 6before/after
Yo-Yo test was significant.
The post-hoc revealed that both pre- and post- Yo-
Yo test values of glucose, creatine-kinase, lactate
dehydrogenase, total cholesterol, triglyceride, and
Table I. Performance measures (i.e., total distance) and peak heart rate (HRpeak) during the Yo-Yo test, RPE scores, and resting core
temperature (mean +s) at 07:00 h (morning) and 17:00 h (evening).
Morning Evening Pvalue
Total distance (m) 1763.64 +482.48 2043.64 +533.5 P¼0.01
HRpeak (beats min
71
) 190.9+5.1 192 +7.3 NS
RPE 14.18 +1.4 14.09 +1.7 NS
Temperature (8C) 36.1 +0.2 36.9 +0.3 P50.0005
4O. Hammouda et al.
Downloaded by [Universite Laval] at 09:37 14 January 2013
high-density lipoprotein were higher in the afternoon
than the morning (P¼0.025 and P50.0005 for
glucose, P¼0.032 and P50.0005 for creatine-
kinase, P¼0.0007 and P50.0005 for lactate dehy-
drogenase, P¼0.0025 and P50.0005 for total
cholesterol, P¼0.0024 and P50.0005 for triglycer-
ide, P¼0.0099 and P50.0005 for high-density
lipoprotein in the morning and the evening respec-
tively). Concerning Lactate levels (Figure1), only
post-exercise values were higher in the evening
(P50.0005) and no significant effect has been
observed for resting values. Moreover, the result
showed a significant correlation between the total
distance covered during the Yo-Yo test and the post-
exercise lactate levels (r¼0.69 and P¼0.017). All
the measured biochemical variables (i.e., triglyceride,
glucose, creatine-kinase, lactate dehydrogenase, and
high-density lipoprotein) were raised after the
exercise with respect to pre-exercise at the two
times-of-day (P50.0005). Significant interactions
time-of-day 6before/after Yo-Yo test were identi-
fied for lactate as well as for creatine-kinase, lactate
dehydrogenase, total cholesterol, triglyceride, and
high-density lipoprotein (Table III), indicating great-
er rises after the evening test. The percentages of
changes between before and after the Yo-Yo test at
the two times-of-day are presented in Table IV.
However, values of uric acid were higher in the
morning before the Yo-Yo test with respect to
evening values (P¼0.0015). The diurnal variations
of uric acid were blunted after the test due to
significant rises of these parameters after the Yo-Yo
test only in the evening (P50.011) (Table IV).
Furthermore, there was a significant time-of-day 6
before/after Yo-Yo test interaction for this parameter
(Table III), indicating that the rise in this marker
after exercise was higher in the evening than the
morning (Table IV).
Discussion
To the best of the authors’ knowledge, this is the first
investigation to study the time-of-day effects on
biochemical responses to intermittent exercise to
exhaustion (i.e., Yo-Yo test). The results confirmed
the diurnal pattern of specific-endurance perfor-
mance in football players. The present study findings
also indicated that biochemical responses show a
diurnal fluctuation during the intermittent endur-
ance exercise.
With regard to performance results, the findings of
the present work indicated that the total distance
during the Yo-Yo test was higher in the evening
session indicating that participants may have had
higher maximal oxygen uptake values at this time
point. In this context, a time-of-day effect on oxygen
uptake has been shown at rest and during submax-
imal exercise, but not on maximal oxygen uptake
(_
VO
2max
) (Brisswalter et al., 2007; Reilly et al.,
1997). Lower oxygen uptake in the morning than the
afternoon is probably due to slower oxygen uptake
kinetics or to a decrease in maximal oxygen uptake in
the morning (Reilly et al., 1997). Moreover, Forsyth
and Reilly (2004) demonstrated the occurrence of
time-of-day effects for oxygen uptake at lactate
threshold. However, several studies of the time-of-
day effects on aerobic metabolism have generally failed
to report diurnal variation in oxygen uptake, especially
with near maximal intensities (Bessot et al., 2006;
Carter et al., 2002; Dalton, McNaughton, & Davoren,
1997; Deschenes et al., 1998). Moreover, the present
study did not show any diurnal change in peak heart
rate during the Yo-Yo test. Nevertheless, previous
studies detected significant diurnal variation in heart
rate during submaximal exercise (Forsyth & Reilly,
2004; Waterhouse et al., 2007). This diurnal variation
of heart rate is most probably essentially due to
circadian variation of core temperature (Waterhouse
et al., 2007). Indeed, many measures of physical
performance display circadian rhythms which are
closely in phase with the variation in body temperature
(Drust, Waterhouse, Atkinson, Edwards, & Reilly,
2005). Alternatively, the lack of diurnal variation of
peak heart rate found in the present study is difficult to
explain.
Regarding the specific aspect of the Yo-Yo test, as
mentioned in the introduction, it has been shown
that professional football players perform at an
optimum between 16:00 h and 20:00 h when not
only football-specific skills but also measures of
Table II. Dietary record of the participants (mean +s).
Nutriments Kilocalorie
CHO
(g)
Protein
(g)
Fat
(g)
CHO
(%)
Protein
(%)
Fat
(%)
Cholesterol
(mg day
71
)
Daily Intake 3304 +706 431.13 +142 103.11 +19 106.58 +62 51.27 +6.1 12.31 +1.1 29.18 +5.3 356.32 +265
Reference Dietary
Intake
(2300–3450)
a
(400–500)
a
(70–110)
a
(100–140)
a
(45–65)
b
(10–30%)
b
(25–35)
b
5350
CHO: Carbohydrate, Vit: Vitamin.
a
Reference dietary intakes for Tunisian adult men (Aounallah-Skhiri et al., 2011).
b
Reference dietary intakes (acceptable macronutrient distribution range) (Otten et al., 2006).
Diurnal variation of biochemical responses in soccer players 5
Downloaded by [Universite Laval] at 09:37 14 January 2013
physical performance are at their peak (Reilly et al.,
2007). However, research about time-of-day effect
on aerobic performance is still inconclusive. The
discrepancies in the findings between the present
results and previous studies might be due to the fact
that participants didn’t satisfy the criteria of maximal
oxygen uptake attainment (i.e., at any time-of-day)
(Drust et al., 2005). Particularly, the Yo-Yo test was
specifically designed to evaluate the ability to per-
form high-intensity intermittent exercise (Krustrup
et al., 2003) and it correlates with match perfor-
mance (Bangsbo et al., 2008). In this context,
maximal values have been reached for aerobic energy
turnover and high anaerobic energy production was
identified toward the end of this test (Krustrup et al.,
2003; Bangsbo et al., 2008).
The results of the present study showed that lactate
and glucose responses to the exercise were signifi-
cantly higher in the evening than the morning which
could indicate a greater anaerobic contribution with
higher mobilisation of glucose metabolism at this
time-of-day. This increase in the lactate levels is in
Table III. Statistical results from analysis of variance and diurnal variations of selected biochemical parameters before and after the Yo-Yo test performed at 07:00 h (morning) and 17:00 h (evening)
(mean +s).
Before exercise After exercise
Morning Evening Morning Evening ANOVA
Mean +sCI Mean +sCI Mean +sCI Mean +sCI TOD before/after YYIRT Interaction
TC (mmol L
71
) 2.81 +0.57 2.4–3.2 3.21 +0.66
$
2.8–3.7 3.35 +0.62
x
2.9–3.8 4+0.7
$,x
3.5–4.5 F
(1.14)
¼47.9; P50.0005 F
(1.14)
¼34.4; P50.0005 F
(1.14)
¼5.6; P¼0.048
HDL (mmol L
71
) 1.02 +0.13 0.9–1.1 1.14 +0.13
$
1.0–1.2 1.19 +0.14
x
1.1–1.3 1.42 +0.12
$,x
1.3–1.5 F
(1.14)
¼41.9; P50.0005 F
(1.14)
¼81.9; P50.0005 F
(1.14)
¼7.1; P¼0.023
Tri (mmol L
71
) 0.96 +0.45 0.7–1.3 1.2 +0.52
$
0.9–1.6 1.32 +0.53
x
1.0–1.7 1.73 +0.63
$,x
1.3–2.2 F
(1.14)
¼18.9; P¼0.0015 F
(1.14)
¼13.9; P¼0.0039 F
(1.14)
¼6.8; P¼0.026
LDH (IU L
71
) 351.64 +24.57 335.1–368.1 396.82 +25.87
$
379.4–414.2 471 +36.1
x
446.7–495.3 542.91 +40
$,x
516.0–569.8 F
(1.14)
¼108.1; P50.0005 F
(1.14)
¼211.8; P50.0005 F
(1.14)
¼6.7; P¼0.027
CPK (IU L
71
) 160.45 +18.68 147.9–173.0 173.73 +14.48
$
164.0–183.5 191.18 +21.13
x
177.0–205.4 219.27 +27.74
$,x
200.6–237.9 F
(1.14)
¼39.6; P50.0005 F
(1.14)
¼150.6; P50.0005 F
(1.14)
¼6.9; P¼0.025
GLC (mmol L
71
) 4.4 +0.15 4.3–4.5 4.6 +0.1
$
4.5–4.7 5.28 +0.29
x
5.1–5.5 5.7 +0.43
$,x
5.4–6.0 F
(1.14)
¼36.8; P50.0005 F
(1.14)
¼132.8; P50.0005 F
(1.14)
¼6.1; P¼0.033
UA (mmol L
71
) 268.25 +29.18
$
248.6–287.9 245.11 +22.4
$
230.0–260.2 317.03 +24.13
x
300.8–333.2 318.57 +13.14
x
309.7–327.4 F
(1.14)
¼5.0; P¼0.049 F
(1.14)
¼346.6; P50.0005 F
(1.14)
¼16.1; P¼0.0025
$
Significant difference between time of test.
x
Significant difference from resting values. CI: confidence interval. TC ¼total cholesterol; HDL ¼high density lipoprotein; Tri ¼triglyceride; LDH ¼lactate
dehydrogenase; CPK ¼creatine phosphate kinase; GLC ¼blood glucose; UA: uric acid; YYIRT ¼Yo-Yo intermittent recovery test.
Table IV. The percentages of changes between before and after the
YYIRT in the morning and the evening.
Morning Evening
Lac (%) 89.37 +3.09 88.71 +3.25
TC (%) 15.81 +8.33 19.59 +11.44
HDL (%) 14.22 +5.14 19.47 +8.82
Tri (%) 25.95 +17.64 29.89 +19.78
LDH (%) 25.12 +5.66 26.68 +5.61
CPK (%) 16.05 +4.36 20.31 +5.08
GLC (%) 16.65 +3.52 18.84 +5.86
UA (%) 15.52 +4.67 23.12 +5.41
Lac ¼lactate level; TC ¼total cholesterol; HDL ¼high density
lipoprotein; Tri ¼triglycer ide; LDH ¼lactate dehydrogenase;
CPK ¼creatine phosphate kinase; GLC ¼blood glucose; UA: uric
acid; YYIRT ¼Yo-Yo intermittent recovery test.
Figure 1. Lac levels measured at 07:00 and 17:00 h before and
after the Yo-Yo test. c: Significant difference between time of test
(P50.001). f: Significant difference from resting values (P5
0.001). Lac ¼lactate.
6O. Hammouda et al.
Downloaded by [Universite Laval] at 09:37 14 January 2013
accordance with previous results during similar
exercises where time-of-day was not investigated
(Krustrup et al., 2003; Rampinini et al., 2010). In
addition, glucose mobilisation has been observed
after maximal aerobic exercise (Marliss et al., 2000).
Moreover, similarly to the present study findings,
diurnal variation of lactate responses has been
identified during mild (Waterhouse et al., 2009),
maximal aerobic (Forsyth & Reilly, 2004, 2005) and
anaerobic (Hammouda et al., 2011; Hammouda,
Chtourou, Chahed, et al., 2012) exercises and would
be indicative of raised anaerobic metabolic activity in
the evening (Thomas & Reilly, 1975). The diurnal
variation of lactate levels could be induced by
catecholamine activity (Deschenes et al., 1998) since
catecholamines, particularly epinephrine, follow very
similar patterns in response to exercise as those
observed for lactate. Indeed, a pronounced circadian
variation in catecholamine activity has been observed
(Akerstedt, 1979) with a peak in the early afternoon
and a trough occurring during the night.
In this context, our group has recently identified
diurnal variations of resting levels of various bio-
chemical markers (i.e., creatine-kinase, lactate dehy-
drogenase, uric acid, glucose, total antioxidant
status) in trained athletes (Hammouda, Chahed,
et al., 2012). These findings could explain the diurnal
pattern of metabolic responses during the Yo-Yo test.
Moreover, Dalton et al. (1997) explained this diurnal
change in lactate, partly, by circadian changes in core
temperature. Indeed, the elevation in body tempera-
ture would increase the activity of enzymes such as
phosphofructokinase and lactate dehydrogenase,
which could in turn increase the blood glucose and
lactate production during exercise and, therefore,
increase absolute performance. In agreement with
this, the results of the present work showed that
lactate dehydrogenase and creatine-kinase increase
during the Yo-Yo test was significantly higher in the
evening. This increase could be indicative of higher
exercise induced muscle damage in the evening
which reflects essentially the higher mechanical and
anaerobic solicitation at this time-of-day.
Similarly to the present study, it has been shown
that lactate concentrations and absolute core tem-
perature at the point of exhaustion were significantly
higher during time to exhaustion in aerobic endur-
ance performed at 18:00 h (Bessot et al., 2006). In
this context, the increase in levels of the selected
parameters observed after the Yo-Yo test seems to be
linked, at least in part, to the total distance of the
exercise, i.e. performance which resulted to be
greater in the evening relative to the morning test
session. Indeed, in agreement with the present study
findings, total distance during the Yo-Yo test has
been shown to be significantly correlated to the rate
of blood lactate accumulations (Krustrup et al.,
2003). However, the increase in the capacity for
anaerobic work during the day as reported in the
literature (Chtourou, Chaouachi, Driss, et al., 2012;
Chtourou, Chaouachi, Hammouda, et al., 2012;
Chtourou, Driss, et al., 2012; Chtourou, Hammou-
da, Chaouachi, et al., 2012; Chtourou, Zarrouk,
et al., 2011; Hammouda, Chtourou, Chahed, et al.,
2012; Hammouda et al., 2011; Souissi et al., 2004)
can, also, explain these results. Accordingly, many
biochemical markers, e.g., transaminase, blood urea,
total bilirubin, total antioxidant status, and homo-
cysteine, including those of the present study,
showed a diurnal pattern during repeated sprints
cycling (Hammouda et al., 2011) and the Wingate
test (Hammouda, Chtourou, Chahed, et al., 2012).
The dietary analyses of macronutrient intakes
indicated that carbohydrate and protein, as well as
fat intakes, are situated in the reference dietary
intakes (Aounallah-Skhiri et al., 2011; Otten et al.,
2006) (Table II), which indicate that the present
findings on biochemical responses are not affected by
dietary nutrient intakes.
This study also shows that plasma levels of uric acid
increased after the exercise in both times-of-day. The
observation that uric acid levels increase in response
to the soccer-specific exercise is consistent with the
findings from another study (Magalha˜es et al., 2010).
This increase could be due to an enhanced contribu-
tion of purine metabolism during the Yo-Yo test.
Indeed, recent data from Krustrup et al. (2006)
showed a significant decrease in players’ muscle
adenosine triphosphate (ATP) levels after an intense
exercise period in the second half and after the entire
football match. Moreover, it is well established that
uric acid is a powerful antioxidant and has a
protective role like vitamins E and C (Hooper, Scott,
& Zborek, 2000). However, the time-of-day effect on
uric acid was suppressed after the exercise. Thus,
maximal exercise seems to suppress diurnal variation
of the antioxidant system.
Additionally, since our group has recently identi-
fied diurnal variations in resting levels of various
biochemical markers in football players (Hammou-
da, Chahed, et al., 2012), the post-exercise levels of
the selected parameters could be influenced by
resting levels.
Alternatively, the present results showed that levels
of total cholesterol, triglyceride, and high-density
lipoprotein increased significantly after the exercise
indicating an enhanced lipid mobilisation. In this
context, the lipids parameter mobilisation after the
exercise could be explained by the fact that aerobic
energy turnover is highly recruited during this test
(Krustrup et al., 2003). Moreover, inspection of the
data reveals that maximal exercise evokes a chron-
obiological effect on variables of lipid profile. Indeed,
levels of the selected markers of lipid profile are
Diurnal variation of biochemical responses in soccer players 7
Downloaded by [Universite Laval] at 09:37 14 January 2013
higher in the evening session. These findings could
be explained by the higher resting values of the
selected parameters observed in the evening. Simi-
larly, previous researches reported that free and
esterified cholesterol of low-density lipoprotein and
high-density lipoprotein showed a significant diurnal
variation: lowest values were seen early in the
morning followed by an increase before breakfast,
with the highest levels during the afternoon in most
cases (Kessler Celia, Van Cauter, & Schoeller,
1995a; Miettinen, 1980). Moreover, daily changes
in triglyceride concentrations are also influenced by
meal intake (Kessler et al., 1995b). Indeed, when
mixed meals are eaten according to a usual eating
schedule (three daily meals), plasma triglyceride was
shown to increase progressively from early morning
to midnight (Rivera-Coll, Xavier, & Antoni, 1994).
In this context, it has been indicated that postpran-
dial lipid, lipoprotein, and apolipoprotein concentra-
tions are affected by circadian factors (Romon et al.,
1997).
A disadvantage in the evaluation of plasma
triglyceride is its high intra-and inter-individual
variability (Bookstein, Gidding, Donovan, & Smith,
1990). In contrast to plasma cholesterol, plasma
triglyceride is highly variable during the day due to
food intake (Kuo & Carson, 1959). As humans are at
most parts of the day in a postprandial state,
measuring plasma triglyceride in the fasting state
underestimates the total diurnal triglyceride load.
A limitation of this study is that oral temperature
was not measured after exercise to estimate the rate of
change with exercise at the different times-of-day.
Moreover, performance during the Yo-Yo test was
recorded only at two times-of-day. Therefore, further
studies should investigate the time-of-day effects on
biochemical responses and use more time points (i.e.,
circadian rhythm). Moreover, biochemical para-
meters were recorded only before and after the Yo-
Yo test. Therefore, further studies should investigate
a delayed blood sample to check peak values.
In conclusion, this investigation demonstrated the
occurrence of time-of-day effects for specific endur-
ance performance and biochemical responses in foot-
ball players. The principal results of this study show
that all the selected biochemical variables (i.e., lipid
profile, glucose, and enzymes) are higher in the evening
than the morning exercises. In fact, the higher
performance recorded in the evening during the
Yo-Yo test could be due to higher metabolic responses
at this time-of-day. Therefore, the higher biochemical
responses observed in the evening could explain, in
part, the greater performance and metabolic solicita-
tion at this time-of-day. Moreover, soccer-specific
endurance performances as well as biochemical res-
ponse measures should be carried out at the same time-
of-day to minimise circadian influences.
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