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Caffeine Ingestion Reverses the Circadian Rhythm Effects
on Neuromuscular Performance in Highly Resistance-
Trained Men
Ricardo Mora-Rodrı
´guez*, Jesu
´s Garcı
´a Pallare
´s, A
´lvaro Lo
´pez-Samanes, Juan Fernando Ortega,
Valentı
´n E. Ferna
´ndez-Elı
´as
Exercise Physiology Laboratory, University of Castilla-La Mancha, Toledo, Spain
Abstract
Purpose:
To investigate whether caffeine ingestion counteracts the morning reduction in neuromuscular performance
associated with the circadian rhythm pattern.
Methods:
Twelve highly resistance-trained men underwent a battery of neuromuscular tests under three different
conditions; i) morning (10:00 a.m.) with caffeine ingestion (i.e., 3 mg kg
21
;AM
CAFF
trial); ii) morning (10:00 a.m.) with
placebo ingestion (AM
PLAC
trial); and iii) afternoon (18:00 p.m.) with placebo ingestion (PM
PLAC
trial). A randomized, double-
blind, crossover, placebo controlled experimental design was used, with all subjects serving as their own controls. The
neuromuscular test battery consisted in the measurement of bar displacement velocity during free-weight full-squat (SQ)
and bench press (BP) exercises against loads that elicit maximum strength (75% 1RM load) and muscle power adaptations
(1 m s
21
load). Isometric maximum voluntary contraction (MVC
LEG
) and isometric electrically evoked strength of the right
knee (EVOK
LEG
) were measured to identify caffeine’s action mechanisms. Steroid hormone levels (serum testosterone,
cortisol and growth hormone) were evaluated at the beginning of each trial (PRE). In addition, plasma norepinephrine (NE)
and epinephrine were measured PRE and at the end of each trial following a standardized intense (85% 1RM) 6 repetitions
bout of SQ (POST).
Results:
In the PM
PLAC
trial, dynamic muscle strength and power output were significantly enhanced compared with AM
PLAC
treatment (3.0%–7.5%; p#0.05). During AM
CAFF
trial, muscle strength and power output increased above AM
PLAC
levels
(4.6%–5.7%; p#0.05) except for BP velocity with 1 m s
21
load (p = 0.06). During AM
CAFF
, EVOK
LEG
and NE (a surrogate of
maximal muscle sympathetic nerve activation) were increased above AM
PLAC
trial (14.6% and 96.8% respectively; p#0.05).
Conclusions:
These results indicate that caffeine ingestion reverses the morning neuromuscular declines in highly
resistance-trained men, raising performance to the levels of the afternoon trial. Our electrical stimulation data, along with
the NE values, suggest that caffeine increases neuromuscular performance having a direct effect in the muscle.
Citation: Mora-Rodrı
´guez R, Pallare
´sJG,Lo
´pez-Samanes A
´, Ortega JF, Ferna
´ndez-Elı
´as VE (2012) Caffeine Ingestion Reverses the Circadian Rhythm Effects on
Neuromuscular Performance in Highly Resistance-Trained Men. PLoS ONE 7(4): e33807. doi:10.1371/journal.pone.0033807
Editor: Reury F. P. Bacurau, University of Sao Paulo, Brazil
Received January 2, 2012; Accepted February 22, 2012; Published April 4, 2012
Copyright: ß2012 Mora-Rodrı
´guez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Ricardo.mora@uclm.es
Introduction
Athletes’ performance is diminished in the early morning and
late night in comparison to the afternoon and evenings [1–4]. This
time-of-day effect on performance occurs during short-term
competition events that rely on muscle strength and power output
[1,5], as well as during long term endurance events [6,7]. The
morning reductions in performance can be observed during simple
continuous motor tasks (e.g., pedalling [8] or swimming [9]), as
well as during complex motor control tasks that involve integration
of information (e.g., tennis serve [10] or handwriting [11]). Even
though the relationship between the circadian rhythm pattern and
the declines in sport performance is well known, the underlying
causes for this diminished motor performance are far from being
established. This endogenous clock involves variations in basal
body temperature and blood concentration of hormones, which in
turn could affect body fluids, urinary metabolites excretion and
cardiac response (i.e., basal heart rate and blood pressure)
[3,12,13]. Alternatively, circadian rhythm may affect performance
by altering actin-myosin cross bridging processes [14], phosphagen
metabolism and/or muscle buffering capacity [12].
To date, studies have mostly described the relationship between
these circadian rhythm factors and motor performance. Only a
few studies have actually manipulated some of the factors involved
in circadian rhythm (e.g., body temperature) to measure variations
in neuromuscular performance [15]. This research group found
that passive heating somewhat improved morning muscle strength
but still it did not reach the neuromuscular performance level
found in the afternoon. Likewise, active warm-up, despite raising
the morning aural body temperature and increasing muscle force
PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e33807
and power output levels, did not increase motor performance to
the levels found in the afternoon [4,16,17]. These studies suggest
that body temperature is one of the most critical components of
the circadian rhythm effects on motor performance.
Caffeine is an ergogenic aid commonly used by elite athletes
[18], especially since its exclusion from the World Anti-Doping
Agency (WADA) prohibited substances list in 2004. This
adenosine receptor inhibitor crosses the blood brain barrier and
enhances motor performance by maintaining neuro-excitability in
the central nervous system of rats [19]. In humans, caffeine
enhances muscular endurance [20] and maintains muscle strength
after prolonged exercise [21], probably counteracting central
nervous system fatigue. Caffeine not only produces an inhibition of
phosphodiesterase actions [22,23] but at ergogenic doses, it could
also increase calcium mobilization from the sarcoplasmic reticu-
lum [23]. Studies in tetraplegic participants show that caffeine
ingestion delays fatigue by 6% when their paralyzed limbs are
electro-stimulated [24]. In addition, caffeine potentiates contrac-
tion force when ingested at physiological doses during low-
frequency electro-stimulation that produces fatigue [25]. This
suggests that caffeine may also have a direct effect on the neuro-
muscular junction or in the contractile apparatus itself, since
central command is not a factor during electro-stimulation.
There is good evidence that caffeine can improve sprint
performance for highly experienced athletes [26,27,28] or
physically active men [29]. In resistance-trained athletes, muscle
strength measured as one-repetition maximum (1RM) is not
commonly affected by caffeine ingestion [30,31]. However, fatigue
during repeated contraction at submaximal loads (70% of 1 RM)
seems to be delayed by caffeine ingestion although only in lower
body musculature [32]. Testing the neuromuscular effects of
caffeine ingestion using the 1RM load may be not totally
adequate. During the 1RM execution, the time of force
application is relatively long and contraction velocity is normally
below 0.4 m s
21
[33,34], far from most velocities found during
sport actions. Caffeine may enhance contraction velocity and/or
motor unit activation against moderate-low resistances for which
1RM may not be a sensitive test to detect it. To our knowledge, no
study has addressed whether acute caffeine ingestion could be an
ergogenic aid for neuromuscular performance in upper and lower
body musculature when performance is measured using submax-
imal loads that permit high contraction velocity and thus muscle
peak power to be reached.
Due to caffeine effects at different loci (i.e., central nervous
system, adenosine receptors, Na
+
/K
+
ATPase activity, intracellu-
lar calcium and/or plasma catecholamines concentration [27]),
caffeine is likely an effective ergogenic aid to counteract the time-
of-day reductions in motor performance. The objective of this
study was to deliver caffeine orally at ergogenic doses and observe
if it could counter the muscle strength and mechanical power
output reductions observed in the morning. Our hypothesis was
that caffeine ingestion will increase morning neuromuscular
performance in both upper and lower body muscle groups. We
hypothesized that caffeine may fully reinstate the level of muscle
strength and mechanical power output to the levels found in the
afternoon. Finally we hypothesized that the effects of caffeine
would be present without affecting basal temperature or the
hormonal anabolic blood milieu.
Methods
Subjects
Twelve highly resistance-trained men volunteered to participate
in this study (age 19.762.8 yr, body mass 74.662.3 kg, height
173.964.8 cm, body fat 11.660.8%, resistance training experi-
ence 7.262.4 yr). Their one-repetition maximum strength (1RM)
normalized per kg of body mass was 1.1560.08 for the bench
press (BP) and 1.4660.15 for the full-squat (SQ) exercises. The
subjects were informed in detail about the experimental
procedures and the possible risks and benefits of the project.
The study complied with the Declaration of Helsinki, was
approved by the Bioethics Commission of the University of
Murcia, and written informed consent was obtained from each
athlete or from their parents prior to participation. Subjects were
informed that they could resign from participation at any time
during the study. All subjects were light caffeine consumers
(#60 mg d
21
from caffeinated soda or lyophilized coffee in milk).
Experimental design
A randomized, double-blind, crossover, placebo controlled
experimental design was used, with all subjects serving as their
own controls. Participants underwent the same battery of
neuromuscular and biochemical assessments under three different
conditions: i) morning (10:00 a.m.) with caffeine ingestion (i.e.,
3mgkg
21
;AM
CAFF
trial); ii) morning (10:00 a.m.) with placebo
ingestion (AM
PLAC
trial); and iii) afternoon (18:00 p.m.) with
placebo ingestion (PM
PLAC
trial). Trials were separated by 24 to
36 hours in between. The experimental trials were designed to
evaluate the main effects of the time-of-day (morning vs.
afternoon) and caffeine ingestion (0 vs. 3 mg kg
21
) on neuromus-
cular performance and hormonal responses. We selected those
times of day for testing (i.e., 10:00 in the morning and 18:00 in the
afternoon) since they are common training schedules for this group
of elite athletes that usually perform two-a-day practices.
In the caffeine ingestion treatment (AM
CAFF
), caffeine (Durvi-
tan, Seid, Spain) was provided in gelatin capsules filled to deliver a
dose of 3 mg kg
21
body mass. The capsules were ingested 60 min
before the testing protocol because it has been repeatedly reported
that blood caffeine concentration peaks 30–60 min after ingestion
[35,36]. In trials without caffeine ingestion (AM
PLAC
and
PM
PLAC
), the subjects ingested placebo capsules filled with the
same amount of dextrose to avoid identification. The amount of
additional energy provided by the dextrose (,2 kcal) was deemed
negligible.
All subjects had previously participated in experiments
involving the measurement of most of the neuromuscular
assessments performed in this study. Nevertheless, participants
underwent three familiarization sessions before the start of the
experimental trials to avoid the bias of progressive learning on test
reliability (one session in the morning (i.e., 10:00 a.m.) and two in
the afternoon (i.e., 18:00 p.m.)). The last familiarization session,
performed in the morning (10:00 a.m.) of the third day prior to the
beginning of each experiment, included the determination of the
following variables for each subject (described later in detail): 1) the
individual load (kg) that maximizes the muscle power output in the
free-weight SQ and BP exercises; 2) the individual load (kg)
corresponding to 75% of 1RM in both exercises, and; 3) the
intensity of stimuli (i.e., amperage) for electrical stimulation of the
quadriceps that elicited an evoked force of 60% of maximal
isometric strength.
Experimental protocol
The day before and during the three days that the experiment
lasted, the subjects stayed in the sports research center where they
slept and ate all meals. They consumed a diet of 2,800–
3,000 kcal?day
21
, composed of 55% energy intake from carbo-
hydrates, 25% from fat and 20% from protein, evenly distributed
across three meals each day (breakfast at 8:30 a.m., lunch at 13:30
Caffeine Enhances Morning Muscle Performance
PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e33807
p.m. and dinner at 20:00 p. m.).. Subjects refrained from physical
activity other than that required by the experimental trials and
withdrew from alcohol, tobacco and any kind of caffeine intake 4
days before testing. The day before the onset of the experiment,
height was measured to the nearest 0.5 cm during a maximal
inhalation using a wall-mounted stadiometer (Seca 202, Seca Ltd.,
Hamburg, Germany). In every trial, upon arrival to the testing
facility, the subjects’ body weights were determined and body
water estimated in a fasted state using a 4-contact electrode body
composition bio-impedance analyzer (Tanita TBF-300A, Tanita
Corp., Tokyo, Japan). Following, tympanic temperature (Thermo-
scan, Braun, Germany) was measured in triplicate after removal of
earwax when needed. Next, subjects lay supine for 15 min after
which a 9 mL blood sample was withdrawn from an antecubital
vein without stasis. A small portion of the whole blood was used to
determine hematocrit by triplicate using no-heparinized capillary
tubes (70 mL; Hirschmann Laborgerate; Germany) and a micro-
centrifuge (Biocen, Arlesa, Spain). The serum and plasma
obtained after centrifugation (i.e., 3000 g) was immediately stored
at 270uC. Next, the subjects filled out a questionnaire geared to
address whether side effects of caffeine were present [18,37].
Then, subjects ingested the capsules containing either their
individualized caffeine dose (3 mg kg
21
) or the placebo with
330 mL of a fruit milkshake (168 kcals) and a pastry (456 kcals)
that served as a standardized breakfast in the AM trials or as an
afternoon snack in the PM trial (total of 624 kcals and 68 g of
carbohydrate).
After a standardized warm-up that consisted of 5 min of jogging
at 10 km h
21
and 5 min of static stretches and joint mobilization
exercises, the subjects entered the gym to start the neuromuscular
test battery assessments under a strict paced schedule (see Figure 1).
These tests consisted of maximum isometric strength as well as the
measurement of bar displacement velocity for loads that elicit
maximum muscle strength and power output adaptations for
upper and lower muscle groups. The battery ended with 1 set of 6
repetitions of full squat exercise with a load of 85% 1RM. This test
was designed to elicit a high level of sympathetic stimulation [38].
Each of these tests took the subjects between 8 and 15 seconds to
perform. Immediately after this bout, with the subject lying supine,
a second antecubital blood sample (9 mL) was rapidly withdrawn.
Blood hematocrit and hormone levels (i.e., serum testosterone,
cortisol, growth hormone, and plasma nor-and-epinephrine) were
evaluated at the beginning of each trial (PRE) and catecholamines
at the end of the maximal sympathetic stimulation bout of exercise
(POST). Upon completion of the test battery (i.e., ,90 min from
the beginning of the neuromuscular assessments) subjects were
discharged and reminded about their schedule for the next trial.
Maximum dynamic strength and maximal power loads
determination
During the last familiarization session the individual loads that
elicited a bar displacement of 1.00 m s
21
and the load of 75% of
1RM for SQ and BP exercises were identified in a graded loading
test using a linear encoder and its associate software (T-Force
System, Ergotech, Murcia, Spain, 0.25% accuracy). Loads which
elicit a velocity of ,1.00 m s
21
are very close to those that
maximize the mechanical power output for isoinertial upper and
lower-body multijoint resistance exercises (e.g., free-weight squat
or bench press) [33,39]. In turn, 75% of 1 RM has been
described as the minimal load that allows positive adaptations for
maximum strength development in highly resistance-trained
athletes [40–42]. After those loads were individually determined,
changes in bar displacement velocity during SQ and BP exercise
as a consequence of our treatments (i.e., time-of- day and caffeine
ingestion) were measured. Detailed description of the BP and SQ
execution technique, as well as the validity and reliability data of
the dynamic measurement system (ICC = 1.00; CV = 0.57%)
have recently been reported [39] in highly resistance-trained
individuals.
Upper and lower body maximum isometric strength
Maximal isometric voluntary contraction strength on the right
knee (MVC
LEG
) was measured as previously described [21] with a
validity and intra-day reliability of 0.89 for ICC and 3.4% for CV.
Briefly, the subjects sat upright in an adjustable chair with their
arms crossed over their chest and fully fastened to prevent
extraneous body movements. With the right hip and knee flexed at
90uthe right ankle was anchored above the malleolus by a strap
connected to a strain gauge dynamometer (Tedea Huntleigh 1263,
Germany) interfaced with an A/D board (Powerlab 8SP, ADI)
and its related software. Maximal isometric leg strength was
collected in triplicate and the best two performances were
averaged and recorded for statistical analysis. In turn, subject’s
arm maximal isometric voluntary contraction strength (MVC
ARM
)
was measured in the right hand using a calibrated handgrip
dynamometer (Takei 5101, Tokyo, Japan). Participants sat with
0 degrees of shoulder flexion, 90 degrees of elbow flexion and the
forearm and hand in a supine position. The best performance out
of two repetitions (spaced by 2 min recovery) was recorded for
subsequent analysis. The ICC and CV of this measurement were
0.99 and 4.1%, respectively.
Electrically evoked muscle response
Using the same setting previously described for the MVC
LEG
testing, the electrically evoked maximal isometric contraction
(EVOK
LEG
) of the right knee extensors was measured. Electrical
stimulation of the muscle was performed using a four channel
high-voltage stimulator (400 V, Megasonic 313, Medicarin,
Spain). The stimulus was delivered via three pairs of adhesive
patch gel electrodes (4.564.5 cm, Medicarin, Spain) placed on the
skin above the proximal and distal portions of the vastus lateralis,
vastus medialis, and rectus femoris. During preparation for this
test, the intensity of stimuli (i.e., amperage; 33–93 mA at each pair
of electrodes) was raised progressively until it evoked at least 60%
of the participant’s MVC [43]. After 4 minutes of rest, we
delivered two pairs of trains of 500 ms in duration and 20 Hz in
frequency to measure peak evoked force (EVOK
LEG
) at the
transducer (expressed in Kg). We chose those electrical stimulation
parameters because we have found them to be highly reliable in
previous studies from our lab (intra-day CV of 3.5% and intra-day
CV of 5.3%; [44]. Because electrodes were removed after each
trial, their placing was marked with an antiallergenic permanent
marking pen to ensure consistent positioning among trials.
Maximal sympathetic stimulation bout of exercise
At the end of the test battery in each trial, subjects underwent 1
set of 6 free-weight full squat repetitions at 85% of 1 RM to elicit a
high level of sympathetic stimulation [38]. Immediately after-
wards, they lay supine and a 9 mL blood sample was rapidly
withdrawn. Four milliliters of this blood were mixed in a tube
containing 0.4 mL of a solution of reduced glutathione (4.5 mg),
sodium heparin (50 IU), and 20 mL of 0.24 M EGTA for
determination of plasma epinephrine (E) and norepinephrine
(NE) concentration (HPLC with electrochemical detection).
Plasma E and NE concentrations were used as an index of whole
body sympathetic nerve activation [45].
Caffeine Enhances Morning Muscle Performance
PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e33807
Blood concentration of steroid hormones
A portion of the blood was allowed to clot into serum tubes (Z
Serum Sep Clot Activator VacuetteH, Greiner Bio-One GmbH,
Austria) and then spun at 2000 gfor 10 min in a refrigerated (4uC)
centrifuge (MPW-350R, Med. Instruments, Poland) to separate
the serum portion. Serum blood was used to determine total
testosterone (TT), cortisol (C) and growth hormone (GH)
Figure 1. Experimental Protocol. Twelve highly resistance-trained men, in a randomized, double-blind and placebo controlled experimental
design, underwent a battery of neuromuscular and biochemical assessments under three different conditions; i) morning (10:00a.m.) with caffeine
ingestion (i.e., 3 mg kg
21
;AM
CAFF
trial); ii) morning (10:00a.m.) with placebo ingestion (AM
PLAC
trial); and iii) afternoon (18:00p.m.) with placebo
ingestion (PM
PLAC
trial).
doi:10.1371/journal.pone.0033807.g001
Caffeine Enhances Morning Muscle Performance
PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e33807
concentrations using chemiluminescence with an automated
analyzer. For TT assessment the blood was processed with an
Advia Centaur kit (Bayer Diagnostics, Tarrytown, NY, intra-assay
coefficient variation ,7.7%). For C and GH assessment an
immulite 2000 kit was used (Siemens, Los Angeles, Calif., intra-
assay coefficients of variation #7%).
Statistical Analysis
Standard statistical methods were used for the calculation of
means and standard deviation (SD). Shapiro-Wilk test was used to
assess normal distribution of data. Reported sleep quality, fatigue
and nervousness perceptions in the questionnaires were not
normally distributed and a nonparametric statistical technique
was applied. Differences between treatments were analyzed as
differences between mean values by using Friedman’s two-way
rank test. Neuromuscular and biochemical results were analyzed
using one-way analysis of variance (ANOVA) for repeated
measurements. The Greenhouse-Geisser adjustment for sphericity
was calculated. After a significant F test, pairwise differences were
identified using Tukey’s significance (HSD) post hoc procedure.
The significance level was set at p#0.05. Cohen’s formula for
effect size (ES) was used, and the results were based on the
following criteria; .0.70 large effect; 0.30–0.69 moderate effect;
#0.30 small effect [46].
Results
Pre-testing conditions and caffeine side effects
Before the three battery tests assessments (AM
PLAC
,AM
CAFF
and PM
PLAC
) body mass and hematocrit were not different
although body bio-impedance was lower in the afternoon (18:00
p.m.) compared to both morning trials (p = 0.02; 0.05). The
PM
PLAC
tympanic temperature was significantly elevated when
compared to both morning treatments (i.e., 0.7uC; p = 0.02; 0.01;
Table 1). Twenty four hours after the conclusion of the AM
CAFF
trial, the participants did not report problems to sleep or
differences in fatigue perception or nervousness in comparison to
the AM
PLAC
or PM
PLAC
treatments. However, 8.3% of the
participants reported gastrointestinal problems and 16.6% an
increase in the urinary excretion.
Velocity during loads for maximum dynamic strength
and maximal power output adaptations
Velocity for maximal power load (i.e., loads that elicit a bar
displacement of 1.00 m s
21
) in SQ and BP exercises was
significantly increased in AM
CAFF
and PM
PLAC
treatments when
compared to the AM
PLAC
trial (range of increase of 2.5–7.5%;
p = 0.000–0.002; ES from 0.58 to 2.10), except in the BP exercise
between AM
CAFF
and AM
PLAC
treatments where the difference
did not reach statistical significance (p = 0.06; ES = 0.68). Velocity
for maximum strength loads (i.e., load of 75% 1RM) in SQ and BP
exercises was significantly greater in AM
CAFF
and PM
PLAC
than
during AM
PLAC
trial (range of increase of 4.6–6.9%; p = 0.003–
0.023; ES: from 0.68 to 1.35; Figure 2C and 2D). However, the
velocity during loads for maximal power (1 m s
21
, Figure 2A and
2B) and maximum strength adaptations (75% 1RM, Figure 2C
and 2D) in both exercises were not significantly different between
AM
CAFF
and PM
PLAC
treatments.
Maximal isometric voluntary contraction strength and
electrically evoked muscle response
No significant differences were detected in maximal isometric
voluntary contraction strength of the right knee (MVC
LEG
) among
any treatment (AM
PLAC
,AM
CAFF
and PM
PLAC
). The electrically
evoked right leg muscle strength (EVOK
LEG
) was significantly
higher following AM
CAFF
when compared to the AM
PLAC
trial
(16%, p = 0.05, ES = 2.27). Of note, only 7 out of the 12 subjects
performed this test due to equipment schedule limitations. No
significant differences were observed in the maximum isometric
grip strength (MVC
ARM
; Figure 2F) among trials.
Maximal sympathetic stimulation bout of exercise
The increase in plasma catecholamine concentration (i.e., NE
and E) induced by 1 bout of 6 free-weight squat repetitions not to
failure (85% 1 RM) is shown in Figure 3. Prior to exercise and
caffeine ingestion (i.e., PRE) levels of E were similar among trials
and increased a similar amount after the bout of intense exercise
(2.9, 3.7 and 2.8 fold for AM
PLAC
,AM
CAFF
and PM
PLAC
,
respectively). However, the basal level of NE was higher during the
afternoon trial (PM
PLAC
; p = 0.005) than during AM
PLAC
. After
the bout of 6 intense squat repetitions (i.e., POST), plasma NE
concentration increased 5 fold following the AM
CAFF
trial which
resulted in significant higher levels of NE than in the AM
PLAC
trial
(Figure 3; p = 0.02). This suggests higher whole body sympathetic
nerve activity during the AM
CAFF
trial than in the AM
PLAC
trial.
The final levels of NE following the PM
PLAC
trial were no different
than those reported during the AM
CAFF
trial, but tended to be
higher than those following the AM
PLAC
treatment (p = 0.07).
Blood concentration of steroid hormones
Total testosterone, cortisol and growth hormone serum
concentrations in the PRE situation (before caffeine ingestion)
were not different between the AM
PLAC
and AM
CAFF
trials as
Table 1. Physiological conditions before the treatments.
AM
PLAC
AM
CAFF
PM
PLAC
Tympanic temperature (uC) 35.3 60.6 35.3 60.8 36.0 60.4*{
Body mass (kg) 74.6 62.3 74.8 62.2 75.0 62.1
Body water (%) 48.2 61.3 48.3 61.3 48.8 61.3
Body fat (%) 11.5 60.7 11.7 60.9 11.1 60.7
Impedance (V)484617 482 617 460 613*{
Hematocrit (%) 44.8 60.7 45.0 61.0 44.6 60.9
Data are presented as mean 6SD.
*Significant differences compared to the AM
PLAC
values.
{Significant differences compared to the AM
CAFF
values. p#0.05.
doi:10.1371/journal.pone.0033807.t001
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PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e33807
expected. Testosterone and cortisol levels were lower (p = 0.000–
0.006) in the afternoon trial (PM
PLAC
) compared to the AM
PLAC
and AM
CAFF
trials (Table 2). In contrast, growth hormone tended
to be higher during the PM
PLAC
than in the morning trials, but the
high variability prevented us from finding significant differences.
The ratio testosterone-cortisol tended to be higher in the afternoon
trial compared to both morning trials (AM
PLAC
and AM
CAFF
).
However, this trend did not reach statistical significance (Table 2).
Discussion
The purpose of this study was to determine the possible
interaction between the effects of time-of-day (morning vs.
Figure 2. Effects of circadian rhythm pattern and caffeine ingestion on dynamic and isometric maximum strength and muscle
power values for upper and lower body actions. A) and B) Velocity for maximal power and; C) and D) Velocity for maximum strength loads for
squat and bench press exercises; E) Maximal isometric voluntary contraction strength (MVC
LEG
) and electrically evoked strength (EVOK
LEG
) on the
right knee; F) Maximal isometric grip strength. Trials were conducted in the morning (10:00 am) without (AM
PLAC
) or with caffeine ingestion (i.e.,
3mgkg
21
;AM
CAFF
) and in the afternoon (18:00 pm; PM
PLAC
). Data are means 6SD. *Significant differences compared to the AM
PLAC
values. p#0.05.
doi:10.1371/journal.pone.0033807.g002
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afternoon) and caffeine ingestion on neuromuscular performance
(i.e., dynamic and isometric strength as well as muscle power
output) in the upper and lower body musculature. Specifically, we
sought to investigate if the stimulant actions of caffeine could
reverse the reductions in neuromuscular performance observed in
the morning. In addition, we set to determine if acute ingestion of
caffeine at a dose known to enhance endurance performance (i.e.,
3mgkg
21
) would also increase muscle power output in highly
resistance-trained athletes (i.e., AM
PLAC
vs. AM
CAFF
comparison).
Our data supports that caffeine is an ergogenic aid for muscle
power output in the upper and lower body musculature of
resistance-trained individuals. Furthermore, our results suggest
that caffeine ingestion in the morning restores neuromuscular
performance (muscle strength and power output) of upper and
lower muscle groups to levels found in the afternoon trial (i.e.,
AM
CAFF
vs. PM
PLAC
comparison, Figure 2). We consider that
these two findings have practical applications during resistance
exercise training and performance.
Using our dynamic measurement system attached to a barbell
individually loaded to elicit bar displacements of 1 m s
21
,we
found that caffeine ingestion increased the morning SQ and BP
muscle power output by 2.5–5.7% (AM
CAFF
vs. AM
PLAC
;
Figure 2A and 2B). Furthermore, morning caffeine ingestion
improved the velocity against loads that maximize maximum
strength adaptations by 5.3% and 4.6% in SQ and BP respectively
(AM
CAFF
vs. AM
PLAC
; Figure 2C and 2D). Thus, neuromuscular
performance (i.e., maximum dynamic strength and muscle power
output) improved in a range of 3–6% in the morning after caffeine
ingestion, during the most common exercises used for resistance
training (i.e., BP and SQ). We think that these strength and power
output enhancements induced by 3 mg kg
21
of caffeine ingestion
(a dose achieved with approximately 2.5 espresso coffees for a
75 kg athlete) have the potential to prevent the morning declines
in sport performance, allowing athletes to train and compete at the
level of the evening.
A recent meta-analysis defends the existence of an ergogenic
effect of caffeine ingestion on maximal voluntary strength but only
for knee extensors [16]. In contrast, our data suggest that caffeine
ingestion increased similarly upper and lower body morning
dynamic maximum strength (Figure 2, albeit from a close to
significant finding in BP in panel B). Our subjects were young
(,20 yr), but very experienced resistance-trained individuals
(average 7 yrs of training) that used their upper and lower body
during training and competition. Importantly, we found a caffeine
effect only when the dynamic contraction was used since isometric
leg or arm strength were not improved. Out of the 27 studies,
included in the Warren and co-workers’ meta-analysis, maximum
strength was measured using isometric contractions in 21 of them
(i.e., 78% of the entries). It is then possible that the lack of
ergogenic effect of caffeine in the upper body strength found in the
meta-analysis may be due to the large percentage of isometric
contraction studies to evaluate the ergogenic effects of caffeine
Figure 3. Catecholamine response to a maximal sympathetic
stimulation bout of exercise. Norepinephrine and Epinephrine
changes following a bout of 6 free-weight squats repetitions with a load
of 85% of 1 RM in the morning (10:00 am) without (AM
PLAC
) or with
caffeine ingestion (i.e., 3 mg kg
21
;AM
CAFF
) and in the afternoon (18:00
pm; PM
PLAC
). Data are means 6SD for 12 resistance-trained men.
Plasma norepinephrine concentrations reflect whole body sympathetic
nerve activation. *Significant differences compared to the PRE values of
the same treatment. {Significant differences compared to the PRE
AM
PLAC
values. {Significant differences compared to the POST AM
PLAC
values. p#0.05.
doi:10.1371/journal.pone.0033807.g003
Table 2. Serum blood steroid hormone concentration in the two trials in the morning (9:15 a.m.; AM
PLAC
and AM
CAFF
) before
caffeine was ingested and in the afternoon (17:15 pm; PM
PLAC
).
AM
PLAC
AM
CAFF
PM
PLAC
Growth hormone (nmol?L
21
) 0.49 60.53 0.38 60.23 1.05 61.27
Testosterone (nmol?L
21
) 16.1 65.8 16.1 65.0 9.4 64.4*{
Cortisol (nmol?L
21
)5426120 564 693 249 679*{
T:C 61000 31.3 612.9 29.3 69.4 40.6 623.4
Data are presented as mean 6SD.
*Significant differences compared to the AM
PLAC
values.
{Significant differences compared to the AM
CAFF
values. p#0.05.
doi:10.1371/journal.pone.0033807.t002
Caffeine Enhances Morning Muscle Performance
PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e33807
ingestion. Our data contends that acute caffeine ingestion,
increases maximal voluntary strength and power output in the
upper and lower muscle groups.
Several publications propose that the lower core temperature
during the morning in comparison to the afternoon is one of the
factors influencing the reduced morning performance. In fact,
when body temperature is raised passively by resting in a hot
environment [15], or actively by prolonged exercise (.15 min)
muscle performance increased to levels near the afternoon trials
[6,16,17]. Knowing this literature, we attempted to eliminate the
effect of core temperature by providing a standardized warm-up
lasting 10 min that contained continuous running and calisthenics.
Despite the warm-up, a tympanic temperature difference of 0.7uC
between morning and afternoon persisted as has been described
previously [47]. Of note, the trials AM
PLAC
and AM
CAFF
had the
same basal tympanic temperature (Table 1) despite significantly
different muscle performance (Figure 2). Thus, although part of
the differences between the morning and afternoon performance
could be due to the differences in core temperature, the effects of
caffeine in improving morning muscle performance seems to be
predominant in comparison to the effects of core temperature. It is
possible that the combination of raising morning core temperature
to the afternoon trial levels plus caffeine ingestion could have
resulted in larger gains in muscle strength and power. On the
other hand, the amount and intensity of exercise required to
increase core temperature 0.7uC in our 19uC dry-bulb environ-
ment, would have likely been fatiguing and energy depleting.
Caffeine ingestion in the morning, a nutritional habit usual for
many athletes, probably allows similar enhancement in muscle
strength and power output than warming-up through strenuous
exercise or passive warming.
Morning reductions in muscle contractility (i.e., the strength
divided by the electromyographic activity) suggest that the
morning declines in muscle strength are due to peripheral
modifications and not to changes in central neural command
[15,48]. We attempt to measure if the effects of caffeine on
improving morning performance were also due to a peripheral
muscle factor. We electrically stimulated the right leg to contract
by delivering the same individualized current intensity in all trials
and found a 15% larger increase in force production during
AM
CAFF
vs. the AM
PLAC
trial (Figure 2E). Our electrical
stimulation directly depolarizes the motor units under the skin
[44] and thus the increase in isometric force 60 min after
3mgkg
21
of caffeine ingestion is circumscribed to the muscle
itself. Although an ergogenic effect of caffeine through maintaining
central neural command has been suggested [19,21] a peripheral
effect of caffeine has not been discarded [24,25]. Our electrical
stimulation data suggest that caffeine improves neuromuscular
performance by acting directly upon the muscles and is in
agreement with the meta-analysis of Warren et al. [20], that found
that electrically evoked strength was higher with caffeine ingestion
when expressed as percent of maximal voluntary contraction.
After the battery of neuromuscular tests, subjects were required
to perform a bout of 6 free-weight squat repetitions at 85% of 1
RM with the aim of markedly raising sympathetic nerve activity.
We measure plasma norepinephrine concentration (NE) as our
main index of whole body sympathetic activity since it is derived in
more than 80% from the spillover of the terminal nerve endings of
the motoneurons [45]. An acute bout of resistance exercise has
been shown to increase plasma concentration of NE [49]. The
magnitude of the increase in NE may be dependent upon the force
of muscle contraction, amount of muscle stimulated and volume of
resistance exercise [50,51]. Despite using the same short-bout of
resistance exercise in all three trials, we observed that plasma NE
rose higher when caffeine was ingested (AM
CAFF
; Figure 3),
suggesting a facilitated sympathetic nerve activation. In contrast,
plasma epinephrine concentration, which is mostly derived from
the adrenal medulla, did not show a larger increase after caffeine
ingestion in comparison to the other trials. Although caffeine
ingestion has been consistently reported to raise plasma epineph-
rine during endurance exercise to fatigue [52], a similar increase
during a single bout of resistance exercise not to failure is
unreported. We did not obtain samples during recovery and thus
we cannot discard a delayed increase in plasma epinephrine upon
caffeine ingestion. Our electrical stimulation data in conjunction
with the plasma NE values suggest that caffeine increases muscle
strength and power output through a direct effect in the muscle.
We measured resting serum hormones to have an index of the
anabolic-catabolic balance in relation to the performance
measured (i.e., muscle strength and power output). Albeit
measurements of only resting blood serum hormone concentration
have their limitations, their levels have been extensively reported
in resistance-training research [38]. Like others [53,54], we
observed higher levels of blood testosterone and cortisol in the
morning than in the afternoon (Table 2). However, the ratio
testosterone-to-cortisol (T/C) tended to be higher in the afternoon
(ES = 0.51) coinciding with the higher levels of muscle strength
and power output. In contrast, Teo et al. [5], argue that the T/C
ratio does not vary in accordance with the changes across the day
in muscle strength and power output. We were aware that a
moderately-high carbohydrate diet should be consumed to
maintain validity of any observed changes in the ratio of T/C
[55]. Our subjects consumed 55% of their daily calories in the
form of carbohydrates and in addition we provided a snack prior
to every trial which included 68 g of carbohydrate. Additionally,
we measure growth hormone and found a trend for increased
levels in the afternoon, also suggesting (like with the T/C ratio) a
more favorable anabolic state. In conclusion, our blood hormonal
data suggest that in the afternoon, despite an absolute decrease in
the concentration of free testosterone, the larger decrease in serum
cortisol results in a more favorable anabolic state. The contribu-
tion of this hormonal milieu to the increased muscle performance
observed in the afternoon is currently under investigation.
In summary, the acute ingestion of caffeine (3 mg kg
21
) reverses
the morning reductions in maximum dynamic strength and muscle
power output (2.5–7.0%), increasing muscle performance to the
levels found in the afternoon (PM
PLAC
trial). These caffeine
ergogenic effects seem to occur only in dynamic and not in
isometric muscle contractions, for both the upper and lower body
actions, and are independent of body temperature. Our electrical
stimulation data, in conjunction with the plasma norepinephrine
concentration, suggest that caffeine increases muscle strength and
power through an effect directly in the muscle. In conclusion,
caffeine ingestion in the morning, an ergogenic aid of common use
among athletes, avoids the morning reduction in muscle
performance due to circadian rhythm.
Acknowledgments
We thank the collaboration of Prof. Jose´ Marı
´aLo´pez Gullo´n from the
High-Performance Sports Center and University of Murcia. We also
acknowledge the commitment and dedication to the testing of each of the
12 high performance athletes that participated in this investigation.
Author Contributions
Conceived and designed the experiments: RM-R JGP AL-S JFO VEF-E.
Performed the experiments: RM-R JGP AL-S JFO VEF-E. Analyzed the
data: RM-R JGP AL-S. Contributed reagents/materials/analysis tools:
RM-R. Wrote the paper: RM-R JGP AL-S JFO VEF-E.
Caffeine Enhances Morning Muscle Performance
PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e33807
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