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Twenty-four-hour rhythms of musclestrength with a consideration of somemethodological problemsLeana Gonçalves Araujo a , Jim Waterhouse b , Ben Edwards b ,Eduardo Henrique Rosa Santos c , Sérgio Tufik a & Marco Túlio deMello aa Department of Psychobiology, Federal University of São Paulo,UNIFESP, São Paulo, SP, Brazilb Research Institute for Sport and Exercise Sciences, LiverpoolJohn Moores University, Liverpool, UKc Physical Education Faculty, Federal University of Goiás, Goiania,GO, Brazil

Available online: 24 May 2011

To cite this article: Leana Gonçalves Araujo, Jim Waterhouse, Ben Edwards, Eduardo Henrique RosaSantos, Sérgio Tufik & Marco Túlio de Mello (2011): Twenty-four-hour rhythms of muscle strengthwith a consideration of some methodological problems, Biological Rhythm Research, 42:6, 473-490

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Twenty-four-hour rhythms of muscle strength with a consideration of

some methodological problems

Leana Goncalves Araujoa*, Jim Waterhouseb, Ben Edwardsb, Eduardo HenriqueRosa Santosc, Sergio Tufika and Marco Tulio de Melloa

aDepartment of Psychobiology, Federal University of Sao Paulo, UNIFESP, Sao Paulo, SP,Brazil; bResearch Institute for Sport and Exercise Sciences, Liverpool John Moores University,Liverpool, UK; cPhysical Education Faculty, Federal University of Goias, Goiania, GO, Brazil

(Received 24 September 2010; final version received 26 October 2010)

The aim of the present study was to show the presence of circadian rhythm ofmuscle strength under a standardised protocol with controlled parameters tosupport suitable observation of variability during the day. Eight male volunteerswere evaluated once a week for 6 weeks at six different times. Rectal temperature,peak torque (PT), maximum work and the average power of the flexor andextensor knee muscles in the isokinetic mode, as well as of PT in maximumvoluntary isometric contraction of knee extensors at 608 knee flexion, weremeasured. The present study showed rhythms with a period of 24 hours in someindices of muscle strength performance at both speeds of movement and musclegroups. To our knowledge, this is the first study that has shown the presence ofcircadian rhythm in all speeds of movement and muscle groups tested under strictstandardised protocol.

Keywords: daily rhythm; muscle strength; standardisation; isokinetic movement

1. Introduction

Chronobiological oscillations in human physical performance have implications forsports training and competition and also for the rehabilitation of individuals.Twenty-four-hour rhythms have been demonstrated at rest in metabolic variables(e.g. oxygen consumption and carbon dioxide output), ventilatory and cardior-espiratory responses to exercise (e.g. minute ventilation, heart hate, cardiac output,and blood pressure), thermoregulatory variables (e.g. core and skin temperaturesand blood flow) and hormonal secretion (e.g. cortisol and catecholamines) (Reillyet al. 2000).

Studies in humans on the 24-hour rhythm of isokinetic muscle strength (Cabriet al. 1988, Wyse et al. 1994, Gauthier et al. 1996, 2001, Deschenes et al. 1998,Martin et al. 1999, Callard et al. 2000) show conflicting results, however. Thecontroversy might be accounted for at least partly by methodological factors, such asdifferent experimental conditions, the use of measurements of performance andrhythm markers that are not accurate and reproducible, lack of familiarisation of

*Corresponding author. Email: leanaaraujo@yahoo.com.br

Biological Rhythm Research

Vol. 42, No. 6, December 2011, 473–490

ISSN 0929-1016 print/ISSN 1744-4179 online

� 2011 Taylor & Francis

http://dx.doi.org/10.1080/09291016.2010.537444

http://www.tandfonline.com

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subjects with the test procedure, effects of muscle temperature with warm-up andinter-investigator reliability. Differences might also result from individual variabilityof the subjects regarding chronotype, gender, level of physical conditioning andtraining (Reilly and Bambaeichi 2003).

To reduce such differences, it is vital that sufficient control of the volunteers andprotocol be observed. These controls should include monitoring sleep to establish nosleep deprivation is present; monitoring diet in the hours before the experiment, toensure that no stimulants or depressants of the CNS are taken; prohibition ofexercise prior to testing; completion of sufficient sessions to familiarise volunteerswith each exercise; reproducibility and reliability in the use of the isokineticdynamometer; assessment of all performance measures by the same evaluator;sufficient time to recover between sessions and between the different exercises withineach session; a frequency of sampling of at least once per four hours and performanceof the experiment under the same laboratory conditions and season. With thesestandardisations, it is possible to minimise the ‘‘noise’’ that is present in any study.

However, none of the studies previously carried out (and cited above) haveobserved all these controls, and this lack of detailed comparability between thestudies is likely to have contributed to the lack of agreement between them. Theobjective of the present study was to investigate the influence of time of the day onseveral measures of muscle strength whilst observing a controlled protocol with allthe controls together used separately in the articles cited above. The precautionstaken in the present study, aimed to standardise this protocol, will be stressed in theMaterial and Methods section and further consideration of them will be given in theDiscussion section.

2. Material and methods

2.1. Volunteers

After explanation of the protocol and requirements to the volunteers, they signed aformal Consent Form of Participation. The Committee of Ethics in ResearchInvolving Humans of the university approved the study under process # CEP 0424/06. The study methods follow the recommendations of the Declaration of Helsinki of1975 for investigations with humans, and the standards for chronobiologyresearchers reported by Touitou et al. (2004 and 2006) and Reilly and Bambaeichi(2003). The procedures were conducted during the winter time.

Eight male volunteers, moderately active (as defined by the Short Questionnairefor the Measurement of Habitual Physical Activity; Baecke 1982), aged 27+3.2years, with body mass 74.6+5.3 kg, height 174.6+6.3 cm, body mass index (BMI)24.4+1.9 kg/m2, body fat 18%+6.7% and lean mass 82%+6.7% (both assessed byplexmografia), took part. The exclusion criteria were history of orthopaedic surgery;osteomyoarticular disease or lesion; neurological disorders; excessive sleepiness, asdefined by the Epworth Sleepiness Scale (Johns 1991) and use of any medication.Additionally, volunteers were asked to refrain from sleep deprivation during theperiod of the experiment, sleeping the time necessary to wake up rested and do notwake up with the alarm clock, and had not performed shift work and/or taken atransmeridional trip in the 10 days preceding the beginning of experiment.Volunteers were not of an extreme chronotype – in other words, they were‘‘moderate evening types’’, ‘‘moderate morning types’’ or ‘‘indifferent’’, as measuredby the questionnaire of Horne and Ostberg (1976). The volunteers went through a

474 L.G. Araujo et al.

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clinical examination that included routine blood tests, effort electrocardiogram(ECG) and ECG at rest to make sure they were fit for undertaking physicalexercise.

The volunteers were informed about the standard recommendations regardingthe length and time of sleep. They slept as much as necessary in the night before atest session to feel rested. It was at least six hours for all volunteers, which wasconfirmed by actigraphy and the Karolinska sleep diary (Akerstedt and Gillberg1990). They were allowed to sleep before the 06:00 hours test, but had to remainawake for the 02:00 hours test (Reilly and Down 1986, Atkinson and Reilly 1996).As regards diet, the last meal before each test session was performed 3 hours inadvance which was confirmed by phone call, to prevent post-prandial thermogeniceffects, and they were recommended not to ingest caffeine or alcohol within the 24hours preceding the data collection, which was confirmed by a food diary.Additionally, volunteers were asked to refrain from heavy physical activity duringthe experiment (Atkinson and Reilly 1996).

2.2. Experimental design

2.2.1. Familiarisation sessions

In this phase of the experiment, the volunteers reported to the laboratory at the sametime of the day at weekly intervals on three different occasions (Reilly andBambaeichi 2003) in order to become familiar with the procedures and minimise anylearning effects upon the results (Kannus 1994). The volunteer’s position on theisokinetic dynamometer was recorded and replicated in all subsequent test sessions.

2.2.2. Main investigation

The subjects reported to the laboratory for data collection at six different times(02:00, 06:00, 10:00, 14:00, 18:00 and 22:00 hours), always on the same day of theweek. There was one test session each day and an interval of 7 days between sessions,which was sufficient time for a full recovery from previous test sessions (Harma et al.1982, Reilly and Down 1986), avoid training effect and also prevented interferencedue to changed routines between weekdays and weekends (Folkard and Monk 1980).The Latin-square cross-sectional design also corrected for any ‘‘order effect’’.

The volunteers reported to the laboratory 1 hour before the test sessions. At thestart of the session, volunteers rested for 30 minutes, awake and in the supine position,in order to reduce the influence of any previous physical activity. This precaution wastaken since physical activity is an exogenous factor that might mask the endogenousbiological clock (Edwards et al. 2002). During this time, rectal temperature using arectal esophageal temperature probe (Steri-Probe/Cincinnati Sub-Zero Products,Inc.) (Cincinnati, OH; accuracy�0.188C) was measured by a logger (Mini-mitter Co.,Inc., Bend, OR, USA), with a sampling frequency of one minute. The last five minutesof the recording were used as a measure of resting core temperature.

The volunteers then carried out 30 seconds of active stretching of the musclegroups involved in the tests and then warmed up for five minutes on a cycleergometer at the low intensity of 85 W. This procedure avoided raising their muscleand body temperature to an extent that would mask their performance rhythm(Wyse et al. 1994).

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Volunteers were then positioned and stabilised on the isokinetic dynamometer(Biodex Multi-Joint System 3 Pro; Biodex Medical Systems; Shirley, NY, USA) asestablished during the familiarisation studies (see above), aligning their anatomicalaxis of articulation with the axis of the equipment. At this time also, the equipmentwas calibrated (Keating and Matyas 1996).

During the tests of muscle strength, all measurements were taken from thedominant limb by means of a computer-controlled isokinetic dynamometer, withcompensation for effects of gravity throughout the whole range of motion.Mechanical signals were recorded at a sampling frequency of 100 Hz. For eachcontraction mode and angular speed, the volunteers performed three sub-maximalcontractions in order to become accustomed to the equipment and exercise(Kannus 1994). Peak torque (PT), maximum work (MW) and average power (AP)were measured during three consecutive maximum isokinetic contractions of theflexor and extensor muscles of the knee muscles at speeds of 1.05 and 4.19 rad s71

over a range of movement of 908. Afterwards, the knee joint was positioned at 608of flexion, and volunteers were required to exert a maximum voluntary isometriccontraction (MVIC) of the knee extensors for 10 seconds, when the PT wasmeasured. A visual display of contraction strength was shown on a computerscreen.

Four minutes of rest separated each test. During the contractions, subjects wereasked to cross their arms over their chests. All the measurements were made by thesame researcher to maximise test–retest reliability (Coldwells et al. 1994, Sole et al.2007). The researcher also gave standardised base instructions (e.g. please give amaximum effort when I say go) and oral commands to encourage maximumperformance (e.g. come on, come on, push, push) in the controlled temperature of218C+0.58C (Wilk et al. 1991).

2.3. Data analysis

The software Statistica for Windows was used, and the results are presented asmean+standard error (SE). Body temperature and muscle strength variables wereinvestigated by repeated-measures analysis of variance (ANOVA), using onefactor (six times of collection). In cases where there was statistical significance,Tukey’s post-hoc tests were applied. Single cosinor analysis, setting the value oftau, the period of the rhythm, to 24 hour, was used to determine rhythmparameters (Nelson et al. 1979). The method consists of a least-squares regressionanalysis to obtain the best estimates of a cosine function of the form:

fðtÞ ¼ Me þ A cos ðwt þ FÞ

where f(t) is the value at time t of the function defined by parameters Me (themean level, termed the mesor), A (the amplitude, half the range of oscillation), w(the angular frequency, degrees per unit time, with 3608 representing a completecycle of 24 hours) and F (the time of the maximum of the fitted curve, termedthe acrophase).

The existence of a sinusoidal rhythm with a period of 24 hours was confirmed ifthe amplitude of the fitted rhythm was significantly different from zero. In addition,the mean cosine curve for all subjects (group cosine curve) was estimated. In allstatistical tests, the level of significance was set at 5% (p �0.05).

476 L.G. Araujo et al.

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3. Results

3.1. Rectal temperature

The ANOVA showed a significant effect of time (Table 1). A significant 24-hourrhythm was observed for Tr (p 50.05) with F at 17:48 hours, Me of 37.08C and A of0.418C (Table 2 and Figure 1).

3.2. Isokinetic muscle strength

The ANOVA showed statistically significant time-of-day effects for all thevariables except average extensor power. Lowest values were nearly always at06:00 hours and the highest levels generally fell between 14:00 and 18:00 hours(Table 3). There were significant 24-hour rhythms in maximum concentricvoluntary isokinetic contractions at low speed (1.05 rad s71) for the followingvariables: PT of the knee extensors, MW of the knee extensors, MW of the kneeflexors and AP of the knee flexors. The other indices of concentric muscleperformance (extensor AP and flexor PT) at this speed presented highest values inthe afternoon, but the amplitudes of the 24-hour rhythms were not significant(Table 4 and Figure 2).

With isokinetic knee flexion and extension at 4.19 rad8s71, ANOVA showed asignificant time-of-day effect for all variables except extensor AP and flexor AP ofthe knee (Table 5). The post-hoc analyses showed the lowest levels at 06:00 hoursand the highest levels at 18:00 hours. Variables that showed a 24-hour rhythm werePT of the knee extensors and MW of the knee extensors. The rhythms were notsignificant for the other variables measured (extensor AP, flexor PT and flexor AP)(Table 6 and Figure 3).

3.3. Isometric muscle strength

For the MVIC of the knee extensors until exhaustion, neither ANOVA not cosinoranalysis showed a significant time-of-day effect for PT. However, PT showed a trendtowards significant variability during the 24 hours (p ¼ 0.057), with the highest valueoccurring at 18:00 hours (Tables 7 and 8).

4. Discussion

The most important findings from the present study are as follows: (1) 24-hourrhythmicity was detected at both low and high speeds of movement, but it was moreevident at slow speed; (2) the variable that presented the lowest sensitivity tovariation along the 24 hours was AP and (3) variations with a period of 24 hourswere not observed for all modes of muscle contraction and speeds with concentricmovement.

4.1. Temperature and the sample of volunteers

The results obtained from rectal temperature – chosen because it is considered one ofthe main markers of endogenous circadian rhythms – are in line with resultspresented in the literature (see Waterhouse et al. 2005, for example). Moreover, thecosinor parameters observed in our study (F ¼ 17:48 hours, Me ¼ 37.08C and

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Table

1.

Variationoftemperature

withtimeofday(m

ean+SE).

02:00hours

06:00hours

10:00hours

14:00hours

18:00hours

22:00hours

Fp

Tr(8C)

36.78+0.01

36.58+0.01

36.79+0.01

37.22+0.01

37.41+0.01

37.18+0.01

1291

50.005

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A ¼ 0.418C) were very close to those obtained by Giacomoni et al. (2005) in a malepopulation (F ¼ 17:29 hours, Me ¼ 37.08C and A ¼ 0.288C).

This result supports the view that our volunteers in the present study exhibited atypical diurnal pattern of body temperature, as can the conditions under which thebaseline measurements of core temperature were made. This is an important findingbecause of our over-riding concern regarding the conditions of measurement andchoice of the volunteers, as well as the controversy that exists in the literature due tooverlooking standardisation procedures that are essential in such studies.

4.2. Rhythms of physical performance: standardisation of volunteers and conditions

Up to the present time, only a few studies have analysed the strength of maximumcontraction under isokinetic conditions, and these have yielded conflicting results.Wyse et al. (1994) and Atkinson et al. (1994) detected a clear diurnal variation in thetorque produced by extension and flexion of the knees during concentriccontractions, both studies finding maximum values between 18:00 and 20:00 hours.However, this movement was analysed at only a few times within a period of 24hours, not enough to establish the details of such a rhythm in any detail. Cabri et al.(1988) did measure the variable more frequently but did not identify statisticallysignificant variations in the muscle function over the course of the day; however, theydid observe that the differences of torque and level of fatigue were greater inconcentric than eccentric movements. They also detected more pronounced

Table 2. Parameters of the 24-hour rhythm of rectal temperature (n¼8) (mean+SE or CI).

VariableMesor(SE)

Amplitude(CI)

Amplitude %mesor (CI)

Acrophasehour:

minute (CI)

Significance(populationcosine curve)

Tr (8C) 37.0 (0.13) 0.41 (0.35–0.77) 1.1 (0.9–2.0) 17:48 (16:07–21:36) 50.05

Note: Tr¼rectal temperature; CI¼95% confidence interval.

Figure 1. Measured 24-hour rhythm of rectal temperature (Tr) with best-fitting sinusoidsuperimposed. Values are mean+SE.

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Table

3.

Variationofisokinetic

muscle

strength

at1.05rad8s

71withtimeofday(m

ean+SE).

02:00hours

06:00hours

10:00hours

14:00hours

18:00hours

22:00hours

Fp

PT

Ext(N

8m)

218.7+14.3

199.2+20.7

223.3+15.4

229.4+17.6

229.8+15.0

226.6+

14.6

5.94

50.005

MaxWork

Ext(J)

222.7+13.6

208.7+20.3

233.3+14.0

236.7+14.6

240.3+13.4

234.9+

12.0

4.72

50.005

AvPow

Ext(W

)135.8+10.9

131.9+13.9

138.5+9.8

143.6+10.6

143.9+11.3

144.9+

11.2

1.79

0.13

PT

Flex(N

8m)

111.0+5.9

109.9+6.0

117.7+5.7

123.0+7.1

116.8+7.0

116.7+

5.2

4.5

50.005

MaxWork

Flex(J)

137.9+7.7

129.2+10.3

149.0+9.1

152.4+9.3

145.9+8.8

147.6+

7.6

5.5

50.005

AvPow

Flex(W

)82.0+3.7

82.1+4.7

86.6+4.8

91.7+5.0

87.6+5.6

84.3+

4.4

3.94

50.005

Note:Ext¼

extensor;Flex¼flexor;PT¼peaktorque;

MaxWork¼maxim

um

work;AvPow¼averagepower.

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differences at high speeds. In more recent studies, Deschenes et al. (1998) observedthat the variables regarding maximum concentric muscle performance of the kneemovement, with the exception of fatigue, only demonstrated a 24-hour rhythm athigh speeds, while Gauthier et al. (2001) reported similar diurnal variations of theconcentric elbow flexion at both low and high speeds and observed the samevariations for isometric contractions.

There are several possible reasons for such disparities, and these are related todifferent conditions of measurement. Some of these conditions will now beconsidered with regard to the present and previous studies. It is accepted thatsome of the precautions that we discuss below might have been taken in the studiesthat are cited, even though no mention appears in the published manuscript. It is oursuggestion that, in future, such details be included.

(1) External factors: Even though events that take place regularly aresynchronisers of the endogenous time in living creatures (zeitgebers), theyalso have a direct (masking) effect upon the measured rhythm, as a result ofwhich the timing of the endogenous oscillator is obscured (Minors andWaterhouse 1981). Therefore, when the possible presence of 24-hour rhythmin a given variable is sought, these influences must be strictly controlled.These influences include the environmental conditions as well as ensuringthat the volunteers are in a true ‘‘baseline’’ condition.

(2) Sleep deprivation: Sleep deprivation might temporarily change performancepatterns. Studies have shown that one night of sleep deprivation does notworsen muscle strength, but that two nights of sleep deprivation do causedeterioration (Meney et al. 1998, Goh et al. 2001, Bambaeichi et al. 2005).Therefore, we standardised sleep before the experiments and in the nightbefore the test sessions and also ensured that the volunteers remained awakebefore the test at 02:00 hours, thus preventing sleep inertia (Atkinson andReilly 1996). Additionally, excessive sleepiness and trans-meridian travel inthe 10 days prior to the beginning of the experiment were exclusion criteria(Reilly and Edwards 2007). Among the five previous studies on the effect oftime of day on parameters of muscle strength (Cabri et al. 1988, Wyse et al.

Table 4. Parameters of the 24-hour rhythm of the variables of isokinetic contraction in kneeflexors and extensors at 1.05 rad s71 (n¼8) (mean+SE or CI).

Variable Mesor (SE)Amplitude

(CI)Amplitude %mesor (CI)

Acrophasehour:minute

(CI)Populationcosine curve

PT Ext(N8 m)

221.1 (44.7) 12.0 (8.5–15.5) 5.4 (3.8–7.0) 17:18 (12:18–18:54) 50.01

Max WorkExt (J)

229.3 (39.8) 12.4 (2.9–21.9) 5.4 (1.2–9.5) 16:54 (11:42–19:30) 50.01

Max WorkFlex (J)

143.9 (23.2) 8.6 (0.7–16.6)) 5.6 (0.4–11.5) 15:36 (11:48–19:24) 50.05

Av PowFlex (W)

85.4 (12.2) 5.5 (2.1–9.0) 6.4 (2.4–10.5) 14:04 (10:8–16:28) 50.01

Note: Ext¼extensor; Flex¼flexor; PT¼peak torque; Max Work¼maximum work; Av Pow¼mean power;CI¼95% confidence interval.

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Figure 2. Twenty-four-hour rhythms of isokinetic muscle strength at 1.05 rad s71 with best-fitting sinusoids superimposed. Values are mean+SE.

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Table

5.

Variationofisokinetic

muscle

strength

at4.19rad8s

71withtimeofday(m

ean+SE).

02:00hours

06:00hours

10:00hours

14:00hours

18:00hours

22:00hours

Fp

PT

Ext(N

8m)

150.4+8.1

145.1+9.1

153.5+6.7

152.1+8.1

156.7+8.0

152.3+7.9

3.10

0.02

MaxWork

Ext(J)

167.8

+11.6

161.8

+13.5

172.0

+11.0

171.6+12.1

177.9

+11.0

172.4

+11.8

4.7

50.005

AvPow

Ext(W

)295.6

+24.8

279.7

+30.8

293.4

+21.3

294.3

+25.0

300.9

+32.8

308.7

+29.4

1.27

0.29

PT

Flex(N

8m)

117.9+5.6

110.3+6.1

117.4+6.3

119.1+5.5

120.4+4.9

114.5+5.9

3.65

50.005

MaxWork

Flex(J)

114.9+6.8

106.6+8.5

115.9+8.1

118.2+6.6

116.4+7.6

112.7+6.7

2.53

0.04

AvPow

Flex(W

)186.8

+19.3

174.4

+19.6

183.8

+22.0

189.3

+16.0

179.2

+12.9

187.7

+17.9

0.66

0.65

Note:Ext¼

extensor;Flex¼flexor;PT¼peaktorque;

MaxWork¼maxim

um

work;AvPow¼averagepower.

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1994, Deschenes et al. 1998, Gauthier et al. 2001, Giacomoni et al. 2005),only that of Giacomoni et al. (2005) appeared to adopt sleep alteration as anexclusion criterion. None of the studies appeared to standardise sleep in thenight before the test.

(3) Warm-up and physical activity: Physical exercise raises core and muscletemperatures, and so masks the endogenous component of the rhythm in, forexample, muscle strength (Edwards et al. 2002). Also, resistance exercise

Table 6. Parameters of the 24-hour rhythms of isokinetic contraction in knee flexors andextensors at 4.19 rad s71 (n¼8) (mean+SE or CI).

Variable Mesor (SE)Amplitude

(CI)Amplitude %mesor (CI)

Acrophasehour:minute (CI)

Populationcosine curve

PT Ext(N8 m)

151.6 (22.1) 3.7 (0.5–6.9) 3.0 (0.3–4.5) 17:06 (10:36–19:36) p 50.05

Max WorkExt (J)

170.5 (33.3) 5.5 (2.1–9.0) 3.2 (1.2–5.2) 17:30 (12:48–19:24) p 50.005

Note: Ext¼extensor; PT¼peak torque; Max Work¼maximum work; CI¼95% confidence interval.

Figure 3. Twenty-four-hour rhythms of isokinetic muscle strength at 4.19 rad s71 with best-fitting sinusoids superimposed. Values are mean+SE.

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increases the levels of circulating catecholamines; this rise affects cardiovas-cular variables, making it impossible to detect an endogenous rhythm incardiac frequency and oxygen uptake after a long period of exercise or afterstrenuous exercise (Kraemer et al. 1987). Consequently, the volunteers in thecurrent study were prohibited from exercise during the experiment andremained at rest for 30 minutes before the beginning of the data collection.This minimised the influence of previous physical activity on the rhythm ofcore temperature. With regard to physical exercise within the 24 hours beforea test session, Wyse et al. (1994) and Gauthier et al. (2001) appeared not toimpose this condition, whereas Cabri (1988) and Wyse et al. (1994) appearnot to have required rest immediately before the periods of data collection.The warm-up might also raise muscle and core temperature if it is notperformed at a low intensity; a low-intensity warm-up was incorporated intoour study. Gauthier et al. (2001) and Cabri (1988) do not seem to haveconsidered this aspect of the protocol.

(4) Dietary intake and ambient conditions: Some researchers believe that digestionmight change thermogenic control through an increase in the metabolism andthat, on the other hand, fasting for a long period before exercise might affectperformance (Douglas 2002, Reilly and Bambaeichi 2003). Room tempera-ture might also affect control of core temperature and mask the endogenousrhythm (Racinais et al. 2004). Care about a standardised diet was taken by allresearchers except Wyse et al. (1994); the precaution regarding temperatureof the laboratory appears largely to have been ignored by Wyse et al. (1994),Deschenes et al. (1998) and Giacomoni et al. (2005). The present study wascarried out in the same season, the winter time, as a further guard againstchanges in environmental temperature; this precaution appears to have beenobserved only in the studies of Gauthier et al. (2001) and Giacomoni et al.(2005).

(5) Familiarisation and fatigue: Our study was carefully designed to minimise theeffects of learning and fatigue (sessions at weekly intervals and in a Latin-square design to remove ‘‘order effects’’). In addition, great care was taken to

Table 7. Variable of the maximal isometric contraction in knee extensors depending on timeof day (mean+SE).

02:00hours

06:00hours

10:00hours

14:00hours

18:00hours

22:00hours F p

MVIC PT(N8 m)

279.6(22.3)

268.7(20.2)

277.5(20.9)

292.0(15.8)

294.9(18.9)

287.4(18.4)

2.39 0.057

Note: PT¼peak torque.

Table 8. Parameters of the 24-hour rhythm of maximal isometric contraction in kneeextensors (n¼8) (mean).

Variable Mesor Amplitude Acrophase hour:minute Population cosine curve

Fatigue PT (N m) 283.5 11.7 17:42 p 4 0.05

Note: PT¼peak torque.

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ensure complete familiarity with the experimental design and apparatus.Minimising any learning effects maximises the possibility of detectingrhythmic changes. Gauthier et al. (2001) appear not to have incorporatedthis learning phase into their protocols at all; Wyse et al. (1994) had only onefamiliarisation session, and Cabri (1988) did not describe the number offamiliarisation sessions. In addition, Cabri (1988), Deschenes et al. (1998)and Giacomoni et al. (2005) did not report that volunteers performedcontractions before each test in order to become accustomed to theequipment and exercise (Kannus 1994). The Latin square transverse designwas not present in any previous study reviewed here.

(6) Standardisation and calibration of equipment and advice to volunteers: Studieshave shown inconsistent use of techniques to correct measured torque foreffects of the weight of the limb and gravity. Gravitational forces need to beadded to results for knee flexion and subtracted from those for kneeextension. Failure to use such corrections will decrease the reliability of theresults. Cabri (1988) seems not to have standardised use of the isokineticequipment with regard to the exact positioning of the volunteer on theapparatus. Moreover, in the present study, the volunteers receivedstandardised oral commands that were given by the same experimenter andunder the same experimental circumstances (Wilk et al. 1991); suchstandardisation appears to be new to this field of study.

(7) Frequency of data collection: One of the important aspects in the use of thecosinor analysis is the frequency of data collection. At least six points arerequired to enable reliable results to be obtained from cosinor analysis (Reillyand Bambaeichi 2003), but this did not occur in the studies of Wyse et al.(1994) and Deschenes et al. (1998), two studies which used ANOVA toestablish time-of-day effects but were precluded by lack of data points fromcosinor analysis. In the present study, six equi-spaced time points were used.

(8) Choice of subjects: The conflicting results in the studies are also due to inter-individual differences between subjects, including chronotype, gender,familiarisation and level of physical training (Reilly and Bambaeichi 2003).Chronotype was the criterion not considered in the studies of Cabri (1988),Wyse et al. (1994) and Deschenes et al. (1998).

Given these sources of possible error, it becomes relevant to evaluate previousstudies of the chronobiology of isokinetic muscle strength and to compare them withthe present study in which all precautions and standardisations have been observedin an attempt to remove as much variation as possible and thus guarantee theaccuracy and reliability of the results.

4.3. Muscle strength

4.3.1. Muscle group and speed

The data from the present study show that there were greater 24-hour changes atslow (rather than rapid) speeds of contraction and in extensor (rather than flexor)movements. This last aspect is in agreement with the literature, which reports greater24-hour variations in the knee extensor than flexor muscle group in a sample ofmales (Deschenes et al. 1998, Wyse et al. 2004, Giacomoni et al. 2005). However,

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such variation has been observed more often at a low speed of isokinetic movementand in the flexors when females have been studied (Giacomoni and Garnet 2002,Giacomoni et al. 2005). Contributory factors to these differences between kneeflexors and extensors might be that transverse sections of knee extensors comprisetwice the area of knee flexors, and their insertion onto the bone is further from theknee joint (Smith et al. 1997). Gauthier et al. (1996) proposed that the higher themuscular mass, the more marked was the measured rhythm; Atkinson et al. (1994)supported this idea, stating that the higher the muscular mass, the higher theamplitude of the rhythm. Such views would explain why the rhythm amplitude ishigher in males and why it can be easier to demonstrate rhythms in males than infemales.

Much controversy exists regarding the 24-hour variation of speed of movement.Some researchers (for example, Deschenes et al. 1998, Bambaeichi et al. 2004,Giacomoni et al. 2005) suggest that the variation is speed-dependent. The findings ofthe present study lead us to disagree with that statement, 24-hour variation havingbeen found by us at both speeds of movement. Our results corroborate those ofWyse et al. (1994), who found variation with time of day at both low and high speedsof movement and in both extension and flexion of the knee during concentriccontractions. Nevertheless, this last group did not evaluate performance at asufficient number of times to enable a description of 24-hour rhythmicity.Giacomoni et al. (2005) found rhythmicity in peak extensor torque of malevolunteers only at the high speed of isokinetic movement. Cabri et al. (1988) didnot find 24-hour rhythmicity, but the differences were more pronounced at the highspeed and in the concentric movement when compared with the eccentric one. Inaddition, Deschenes et al. (1998) observed time-of-day oscillations only in kneeextension at a high speed and found no significant rhythms in either extension orflexion at a low speed. Our data corroborate the findings of Gauthier et al. (2001),who detected torque rhythmicity during elbow flexion at all speeds analysed, whichsuggests that the physical human performance is controlled independently of thespeed of movement.

One explanation of all this variability in results is that many physiologicalprocesses change with time of the day and that these processes all contribute tomaximum muscular performance. These physiological processes include centralfactors (command of the central nervous system, alertness, and motivation) andperipheral factors (contractibility), and these might be influenced by hormonal, ionicand temperature variations (Birch and Reilly 2002). All these processes are involvedin neuro-motor control during the production of muscle strength, independent of thespeed of movement or the muscle group involved. Therefore, it can be hypothesisedthat variations in muscle performance will vary during the day in a manner thatdepends upon the type of exercise. However, such a hypothesis requires furtherexperimental testing.

4.3.2. Warm-up masking effect

The present study did not detect 24-hour rhythms in isometric exercise, and thisabsence of rhythmicity is in spite of the fact that rhythms are found in metabolic,ergogenic and vasomotor functions, all of which contribute to muscle performance.The performance of two isokinetic tests before the isometric test might have inducedan additional effect of muscle warm-up, increasing muscle temperature and masking

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any time-of-day effect. This possibility is in agreement with the findings of Reillyand Down (1992), who failed to detect 24-hour variation in the Wingate testperformed after two anaerobic muscle tests. Giacomoni et al. (2005) foundsignificant a 24-hour rhythm only when electrical twitches were superimposed dueto the motivation to make the maximum effort. Further work is required to testthese issues.

4.3.3. Torque, power and work

Our study assessed whether the rhythmicity of PT was similar for MW and AP, aswell as whether it varied with speed of movement. Muscle torque differs from othercomponents of physical performance, which are related more closely to speed(Wilmore and Costill 1994). As far as we are aware, only Deschenes et al. (1998)analysed changes in isokinetic variables other than PT and muscle fatigue over thecourse of the day. PT is believed to be the most precise and reproducible marker ofisokinetic muscle function (Kannus 1994), and this might be the reason whychronobiology studies are limited to the assessment of PT. However, the reliability,validation and reproducibility of the variables measured by the isokineticdynamometer (power and work, for instance) have been clearly established(Piencivero et al. 1997, Patterson and Spivey 1992) and should be emphasised infurther studies.

Deschenes et al. (1998), investigating a single repetition (PT), found time-of-dayeffects on maximal work by knee extensors solely at higher velocities of movement. Asimilar result was found with AP of the extensors at the highest speed. Our resultsshowed rhythmicity in more variables than in Deschenes et al.’s group – includingMW at both slow and fast speeds of movement as well as with extensor and flexormuscle groups. The AP showed statistically significant effects of time of day onlywith flexion at the slow velocity. From these results, we suggest that it is easier todetect 24-hour rhythms in maximal performance, as in PT and maximal work, thanin average performance (AP, for example).

5. Conclusion

In conclusion, the present study showed a significant 24-hour rhythm in slow andfast speeds of movement of knee extensors and flexors, but the rhythms were mostmarked for knee extensors at the slow speed. Comparing these results with thosefrom the literature stresses the importance of methodological issues to guarantee thereliability of results in studies of human performance rhythms. If these precautionsare met by using a standardised protocol, then several components muscle strengthshow rhythmicity with a period of 24 hours. There is also the implication that moredetails of the precautions taken need to be reported.

Understanding rhythmic organisation of the motor system is highly relevant,since the system is widely involved in the control of simple tasks, such as theactivities of daily living (ADLs), and in more complex movements, as in sportsactivities. Knowledge of the time of day when best performance can be obtained(including components of muscle strength, speed, power and coordination) hasimplications for work and daily activities as well as for preparing programmes ofphysical training and rehabilitation. Using a sound methodological design is animportant component of studies designed to obtain such knowledge.

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