Caffeine increases maximal fat oxidation during a graded exercise test: is there a diurnal variation?

Abstract

Background
There is evidence that caffeine increases the maximal fat oxidation rate (MFO) and aerobic capacity, which are known to be lower in the morning than in the afternoon. This paper examines the effect of caffeine intake on the diurnal variation of MFO during a graded exercise test in active men.

Methods
Using a triple-blind, placebo-controlled, crossover experimental design, 15 active caffeine-naïve men (age: 32 ± 7 years) completed a graded exercise test four times at seven-day intervals. The subjects ingested 3 mg/kg of caffeine or a placebo at 8 am in the morning and 5 pm in the afternoon (each subject completed tests under all four conditions in a random order). A graded cycling test was performed. MFO and maximum oxygen uptake (VO 2max ) were measured by indirect calorimetry, and the intensity of exercise that elicited MFO (Fat max ) calculated.

Results
MFO, Fat max and VO 2max were significantly higher in the afternoon than in the morning (all P < 0.05). Compared to the placebo, caffeine increased mean MFO by 10.7% (0.28 ± 0.10 vs. 0.31 ± 0.09 g/min respectively, P < 0.001) in the morning, and by a mean 29.0% (0.31 ± 0.09 vs. 0.40 ± 0.10 g/min, P < 0.001) in the afternoon. Caffeine also increased mean Fat max by 11.1% (36.9 ± 14.4 [placebo] vs. 41.0 ± 13.1%, P = 0.005) in the morning, and by 13.1% (42.0 ± 11.6 vs. 47.5 ± 10.8%, P = 0.008) in the afternoon.

Conclusion
These findings confirm the previously reported diurnal variation in the whole-body fat oxidation rate during graded exercise in active caffeine-naïve men, and indicate that the acute ingestion of 3 mg/kg of caffeine increases MFO, Fat max and VO 2max independent of the time of day.

Trial registration
NCT04320446 . Registered 25 March 2020 - Retrospectively registered

Full text

R E S E A R C H A R T I C L E Open Access
Caffeine increases maximal fat oxidation
during a graded exercise test: is there a
diurnal variation?
Mauricio Ramírez-Maldonado
2
, Lucas Jurado-Fasoli
1,2
, Juan del Coso
3
, Jonatan R. Ruiz
2
and
Francisco J. Amaro-Gahete
1,2*
Abstract
Background: There is evidence that caffeine increases the maximal fat oxidation rate (MFO) and aerobic capacity,
which are known to be lower in the morning than in the afternoon. This paper examines the effect of caffeine
intake on the diurnal variation of MFO during a graded exercise test in active men.
Methods: Using a triple-blind, placebo-controlled, crossover experimental design, 15 active caffeine-naïve men
(age: 32 ± 7 years) completed a graded exercise test four times at seven-day intervals. The subjects ingested 3 mg/
kg of caffeine or a placebo at 8 am in the morning and 5 pm in the afternoon (each subject completed tests under
all four conditions in a random order). A graded cycling test was performed. MFO and maximum oxygen uptake
(VO
2max
) were measured by indirect calorimetry, and the intensity of exercise that elicited MFO (Fat
max
) calculated.
Results: MFO, Fat
max
and VO
2max
were significantly higher in the afternoon than in the morning (all P< 0.05).
Compared to the placebo, caffeine increased mean MFO by 10.7% (0.28 ± 0.10 vs. 0.31 ± 0.09 g/min respectively,
P< 0.001) in the morning, and by a mean 29.0% (0.31 ± 0.09 vs. 0.40 ± 0.10 g/min, P< 0.001) in the afternoon.
Caffeine also increased mean Fat
max
by 11.1% (36.9 ± 14.4 [placebo] vs. 41.0 ± 13.1%, P= 0.005) in the morning, and
by 13.1% (42.0 ± 11.6 vs. 47.5 ± 10.8%, P= 0.008) in the afternoon.
Conclusion: These findings confirm the previously reported diurnal variation in the whole-body fat oxidation rate
during graded exercise in active caffeine-naïve men, and indicate that the acute ingestion of 3 mg/kg of caffeine
increases MFO, Fat
max
and VO
2max
independent of the time of day.
Trial registration: NCT04320446. Registered 25 March 2020 - Retrospectively registered
Keywords: Exercise performance, Body composition, Nutrition, Body weight, Dietary supplement
© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
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data made available in this article, unless otherwise stated in a credit line to the data.
* Correspondence: amarof@ugr.es
1
Department of Physiology. Faculty of Medicine, University of Granada, Av.
Conocimiento S/n, 18011 Granada, Spain
2
PROFITH “PROmoting FITness and Health Through Physical Activity”
Research Group, Department of Physical Education and Sport, Faculty of
Sport Sciences, University of Granada, Granada, Spain
Full list of author information is available at the end of the article
Ramírez-Maldonado et al. Journal of the International Society of Sports Nutrition
(2021) 18:5
https://doi.org/10.1186/s12970-020-00400-6

Introduction
Endurance performance has been traditionally under-
stood as a multifactorial concept in which maximal oxy-
gen uptake (VO
2max
), ventilatory thresholds and
muscular efficiency play important roles [1]. Consider-
ably less attention has been paid, however, to the im-
portance of the management of substrate oxidation
during prolonged exercise and its relationship with en-
durance performance [1]. Metabolic flexibility, known as
the capacity to adapt fuel utilization to substrate avail-
ability, has recently been suggested an additional key fac-
tor affecting performance in endurance disciplines [2].
Given that maximal fat oxidation during a graded exer-
cise test (MFO), and the intensity of exercise that elicits
MFO (Fat
max
), have been recognized as potential deter-
minants of metabolic flexibility during exercise [3,4], it
seems plausible that both MFO and Fat
max
strongly in-
fluence endurance performance. Certainly, higher fat
oxidation rates (at the expense of lower carbohydrate
use) at moderate exercise intensities might help spare
endurance athletes’muscle and liver glycogen stores
during training and competition [5].
In athletes, it is known that endurance performance is
poorer early in the morning and late at night compared
with the afternoon [6], and that MFO and Fat
max
are
higher in the afternoon compared to the morning whether
in non-athlete male students [7], in untrained normal-
weight and obese individuals [8], or in endurance-trained
athletes [9]. The difference has been explained by the
higher body temperature, the enhanced neural activation
and contractile properties of the skeletal muscle, and the
higher plasma catecholamine concentrations found in re-
sponse to exercise in the afternoon compared to the
morning and evening [10,11].
Caffeine is a natural alkaloid used by both endurance
and resistance athletes as an ergogenic aid [12,13]. It
does not appear in the World Anti-Doping Agency’s
2004 list of prohibited substances. Interestingly, the
urine caffeine concentration recorded in doping control
tests, especially for athletes of endurance-based sports,
has increased progressively since it was removed from
the above list [14]. Certainly, low-to-moderate doses of
caffeine (~ 3–9 mg/kg) [15] can increase endurance per-
formance [16] via the induction of significant increases
in VO
2max
, peak pulmonary ventilation, and muscle oxy-
gen saturation during submaximal workloads [17,18]. A
recent study by Gutierrez-Hellín et al. [19] also shows
caffeine ingestion to increase the MFO in healthy sub-
jects of both sexes. Similarly, the ingestion of 5–7 mg/kg
of caffeine during steady-state aerobic exercise seems to
increase the utilization of fat as a fuel in detriment to
the use of carbohydrate [20–22]. Preliminary reports also
suggest that caffeine intake may help counteract the di-
urnal variation observed in exercise performance [6,23–
26]. Mora-Rodríguez et al. [24] reported that the acute
ingestion of caffeine reverses the time-of-day reduction
seen in maximum dynamic strength and muscle power
output in resistance-trained men, while Boyett et al. [23]
report that trained athletes are more likely to obtain er-
gogenic effects from caffeine in the morning than the
evening (at least in terms of cycling performance). It
would be of interest to know whether caffeine attenuates
the diurnal variation seen in both the rate of whole-body
fat oxidation during exercise, and in endurance perform-
ance, and whether caffeine has a synergistic effect with
the already known diurnal variation in energy metabol-
ism. The aim of the present work was, therefore, to in-
vestigate the effect of caffeine intake on the diurnal
variation of MFO and Fat
max
during a graded exercise
test in active men. Based on the available scientific lit-
erature, we hypothesised that: (i) The acute ingestion of
caffeine will increase MFO, Fat
max
and VO
2max
inde-
pendent of the time of day. (ii) There will be a diurnal
variation in MFO, Fat
max
and VO
2max
, with values being
higher in the afternoon than in the morning.
Methods
Subjects
Fifteen active men, aged 32 ± 7 years, volunteered to partici-
pate in the current study (clinicaltrials.gov; NCT04320446).
To be included all subjects had to: (i) have a body mass index
of 18.5–28 kg/m
2
, (ii) be non-smokers, (iii) suffer no disease
that might be aggravated by physical exercise, (iv) take no
medication or drugs, (v) be naive caffeine consumers (< 50
mg/day), (vi) have previous experience in endurance training
(i.e., self-reporting of at least 2 years of endurance training in-
cluding three or more training sessions/week [3.6 ± 0.2 ses-
sions/week]), (vii) be free of any caffeine allergy, and (viii)
have incurred no musculoskeletal injury during the previous
month. All subjects were recruited by social networks and
local media, and they provided oral and written informed
consent before their enrolment. Procedures were performed
in accordance with the latest revised Declaration of Helsinki
(2013). The University of Granada Research Ethics Commit-
tee approved the present project (N° 507/CEIH/2018).
Design and methodology
This study had a triple-blind (i.e. participants, evaluation
staff and statistician), placebo-controlled, crossover ex-
perimental design involving a graded exercise test per-
formed by all subjects on four occasions, with each
occasion separated by 7 days (Fig. 1). They were asked
to maintain their physical activity levels and nutritional
habits during the intervention. Subjects ingested either a
dose of 3 mg/kg anhydrous caffeine in powder form (the
extract of HSN® green coffee beans [Harrison Sport Nu-
trition (HSN) Store, Granada, Spain]) or a 100% pure
microcrystalline cellulose placebo [Acofarma, Madrid,
Ramírez-Maldonado et al. Journal of the International Society of Sports Nutrition (2021) 18:5 Page 2 of 9

Spain]) 30 min before each test. Both supplements were
unflavoured, uncoloured and odourless. The use of the
above-mentioned dose was based on the results of previ-
ous studies reporting caffeine to be effective at increas-
ing fat oxidation during exercise in trained athletes [19].
Both the caffeine and placebo were dissolved in 250 ml
of water and served in opaque, indistinguishable recipi-
ents; the subjects were therefore blind to what they had
received.
The study was performed between June and November
2019. Measurements were conducted between 8 and 11
am (providing MFO-morning, Fat
max
-morning, and
VO
2max
-morning), and between 5 and 8 pm in the after-
noon (providing MFO-afternoon, Fat
max
-afternoon, and
VO
2max
-afternoon). The order of (i) the time of the day
when the exercise tests were performed, and (ii) the ad-
ministration of caffeine or placebo, were randomized
using a function included in MS Excel for Windows®.
However, all subjects were tested under all ingestion/
time-of-day condition combinations.
Before testing began (Day 0), subjects’weight and height
were recorded using a Seca model 799 electronic column
scale and stadiometer (Seca, Hamburg, Germany), and
their body mass index calculated as weight divided by the
square of the height (kg/m
2
). The body weight measured
on this day was used in the dosage calculations for the en-
tire experiment. Subjects were asked to be barefoot and to
wear only light clothing during these measurements. Dual
energy X-ray absorptiometry, performed using a Hologic
Discovery Wii device (Hologic, Bedford, MA, USA), was
conducted to determine subject lean and fat mass (kg). All
subjects also completed the HÖME questionnaire to de-
termine their chronotype (i.e., morningness–eveningness).
They were subsequently categorized as (i) definite evening
type (score range 16–30), moderate evening type (31–41),
neither type (score 42–58), moderate morning type (59–
69) and definite morning type (70–86) [27]. Finally, all
subjects were provided instructions: (i) to avoid moderate
and vigorous physical activity 24 and 48 h respectively be-
fore test days, (ii) to adhere to a standardized, personalized
diet (50% carbohydrates, 30% fat and 20% protein) during
the 24 h before each test day and to keep to the same meal
order independent of the time of the day at which the test
was performed, (iii) to arrive at the laboratory in a motor-
ized vehicle to avoid physical activity, and (iv) to fast for 3
h before arrival. Compliance with these instructions was
checked by self-reported dietary and exercise records.
On test days, a personalized dose of caffeine (3 mg/kg)
or placebo was provided before performing the graded
exercise test - undertaken using a Cardgirus Medical Pro
cycle ergometer (C&G Innovations, Cochin, India) under
controlled environmental conditions (temperature:
ranged from 22 to 24 °C and humidity: ranged from 40
to 50%). After substance intake, subjects rested in the
supine position for 30 min to ensure absorption. There-
after, a submaximal graded exercise test was begun. This
consisted of cycling at 50 W maintaining a cadence of
60–100 rpm for 3 min (warm-up protocol), with subse-
quent 25 W increments of the workload every 3 min
until reaching a respiratory exchange ratio of 1.0 [3,28].
They then rested for 5 min with free access to water be-
fore beginning a maximal graded exercise test to
Fig. 1 Study procedures. Abbreviations: DXA; dual energy X-ray absorptiometry test
Ramírez-Maldonado et al. Journal of the International Society of Sports Nutrition (2021) 18:5 Page 3 of 9

measure their VO
2max
. This began with the same warm-
up protocol, followed by increments of 50 W every mi-
nute until self-reported exhaustion [29]. Indirect calor-
imetry data were registered using a CPX Ultima
CardiO2 breath-by-breath gas analyzer (Medical Graph-
ics Corp., St. Paul, MN, USA). A prevent™metabolic
flow sensor (Medgraphics) fitted to a model 7400 orona-
sal mask (Hans Rudolph Inc., Kansas City, MO, USA)
was used to obtain respiratory data. Simultaneously, a
Polar RS800 heart-rate monitor (Polar Electro Inc.,
Woodbury, NY, USA) was used to monitor the heart
rate during both maximal and submaximal graded exer-
cise. The gas analyzer was calibrated immediately before
each graded exercise, according to the manufacturer’s
recommendations.
Submaximal graded exercise test
The VO
2
and VCO
2
data derived from the last 60 s of
each graded exercise stage were taken into account [30].
Fat oxidation rates were estimated from the stoichiomet-
ric equation of Frayn, assuming urinary nitrogen excre-
tion to be negligible [31]. MFO and Fat
max
were
determined by plotting fat oxidation values (dependent
variable) against the relative exercise intensity (inde-
pendent variable) to construct a third degree polynomial
regression curve for each subject (0,0) from a graphical
depiction of fat oxidation values as a function of exercise
intensity [32].
Maximal graded exercise test
The criteria for deeming VO
2max
to have been reached
were: (i) attaining a steady (increase < 2 ml/kg/min) in
VO
2
despite a further increase in workload, (ii) showing
a maximal heart rate between 10 bpm above and below
the age-predicted maximum [33], and (iii) reaching a re-
spiratory exchange ratio of > 1.1 [34]. When these cri-
teria were not met, peak oxygen consumption was taken
into account (i.e., the highest VO
2
value measured over
the last 60 s of the test).
Statistical analysis
Sample size and power calculations were determined
based on the results of a prior study [9]. We considered
MFO differences between (i) morning vs. afternoon and
(ii) caffeine vs. placebo test in order to assess the sample
size requirements for the two-way analysis of variance
(time-of-the day x substance). As a result, we expected
to detect an effect size of 0.05 g/min considering a type I
error of 0.05 with a statistical power of 0.90 with a mini-
mum of 12 participants. Assuming a maximum loss of
20%, we decided to recruited a total of 15 participants.
The results of every test were blindly introduced into
the SPSS v.22.0 package (IBM Corporation, Pittsburgh,
PA, USA); analyses were also performed blind to
experimental conditions. Visual check histograms, Q-Q
plots and Shapiro-Wilk tests were used to check the
normality of all variables. Since all study outcomes were
normally distributed, parametric tests were used to
examine differences between conditions. Two-way ana-
lysis of variance (time-of-the day x substance) was used
to compare MFO, Fat
max
and VO
2max
under different
study conditions. When a significant F value was ob-
tained, a Bonferroni post hoc analysis was performed to
determine pairwise differences. Additional analyses were
conducted after adjusting for age, chronotype, lean mass
and fat mass. Finally, experimental conditions with a
common characteristic (i.e., morning vs. afternoon, and
caffeine vs. placebo) were grouped to independently cal-
culate the effect of the time of the day and substance
provided on MFO, Fat
max
and VO
2max
using pairwise
tests. Significance was set at P< 0.05. Lastly, we also cal-
culated the standardized effect sizes using Cohen’s d co-
efficients. Graphs were plotted using GraphPad Prism 5
(GraphPad Software, San Diego, CA, USA).
Results
Table 1shows the characteristics of the study partici-
pants. The chronotype was homogeneously distributed
(n= 5 moderate evening type, n= 5 neither type, and
n= 5 moderate morning type).
Time-of-day had a significant effect on MFO (P<
0.01), with the latter always higher (ranging from 10.7 to
29.0%) in the afternoon than in the morning. Compared
to the placebo, caffeine intake increased mean MFO by
10.7% in the morning (0.28 ± 0.10 vs. 0.31 ± 0.09 g/min
respectively, P< 0.001; d = 0.32; Fig. 2) and by 29.0% in
the afternoon (0.31 ± 0.09 and 0.40 ± 0.10 g/min, P<
0.001; d = 0.95; Fig. 2). A significant time-of-the day x
substance interaction was observed in MFO (P< 0.001;
Fig. 2).
Table 1 Characteristics of the study subjects (n= 15)
Age (years) 32.4 ± 7.2
Weight (kg) 79.9 ± 10.7
Height (m) 1.8 ± 0.1
Body mass index (kg/m
2
) 25.6 ± 2.3
Fat mass (%) 18.5 ± 3.9
Lean mass (kg) 61.7 ± 9.0
HÖME questionnaire score
Definitive evening type (n [%]) 0 [0.0]
Moderate evening type (n [%]) 5 [33.3]
Neither type (n [%]) 5 [33.3]
Moderate morning type (n [%]) 5 [33.3]
Definite morning type (n [%]) 0 [0.0]
Values expressed as means ± standard deviation
Ramírez-Maldonado et al. Journal of the International Society of Sports Nutrition (2021) 18:5 Page 4 of 9

Time-of-day also had a significant effect on Fat
max
(all
P< 0.01), which was always higher (ranging from 11.1 to
13.1%) in the afternoon than in the morning. Compared
to the placebo, caffeine intake increased Fat
max
by 11.1%
in the morning (36.9 ± 14.4 vs. 41.0 ± 13.1% respectively;
d = 0.30; Fig. 3) and by 13.1% in the afternoon (42.0 ±
11.6 vs. 47.5 ± 10.8%, respectively; d = 0.49; Fig. 3). A
strong trend toward significance time-of-the day x
substance interaction was observed in Fat
max
(P= 0.058;
Fig. 3).
Finally, time-of-day had a significant effect on VO
2max
(P< 0.05), which was always higher (ranging from 3.2 to
3.9%) in the afternoon than in the morning. Compared
to the placebo, caffeine intake increased VO
2max
by 3.9%
in the morning (43.7 ± 7.8 vs. 46.7 ± 7.0 ml/kg/min, re-
spectively; d = 0.40; Fig. 4) and by 3.2% in the afternoon
Fig. 2 Maximal fat oxidation (MFO) in the morning, and in the afternoon, after ingestion of caffeine or the placebo. Panel a: Individual observations for
each subject (grey lines), and the mean for all subjects (black line). Panel b: Individual observations for each subject (black dots), standard deviation
and minimum/maximum values (box-and-whisker plots), and the Pvalue obtained by two-way ANOVA. Similar letters (i.e. a-a; b-b, etc.) indicate
significant post hoc differences
Fig. 3 Intensity of exercise eliciting maximal fat oxidation (Fat
max
) in the morning, and in the afternoon, following the ingestion of caffeine or the
placebo. Panel a: Individual observations for each subject (grey lines), and the mean for all subjects (black line). Panel b: Individual observations
for each subject (black dots), standard deviation and minimum/maximum values (box-and-whisker plot), and the Pvalue obtained by two-way
ANOVA. Similar letters (i.e., a-a; b-b, etc) indicate significant post hoc differences
Ramírez-Maldonado et al. Journal of the International Society of Sports Nutrition (2021) 18:5 Page 5 of 9

(45.4 ± 8.0 vs. 48.2 ± 7.0 ml/kg/min; d = 0.37; Fig. 4). No
significant time-of-the day x substance interaction was
observed in VO
2max
(P> 0.7; Fig. 4).
All the significant differences reported above persisted
after adjusting for age, chronotype, lean mass and fat
mass (data not shown).
Discussion
The present results indicate that caffeine intake increases
MFO and Fat
max
as well as VO
2max
independent of the
time of day. The highest values for these variables were all
obtained in the afternoon after caffeine intake. The results
also show that, in the morning, the values of MFO after
caffeine ingestion are nearly equivalent to those recorded
in afternoon tests without caffeine supplementation. This
suggests that caffeine increases whole-body fat oxidation
during graded exercise in the morning to a value similar
to that seen without caffeine in the afternoon. Overall,
these results suggest that a combination of acute caffeine
intake and exercise at moderate intensity in the afternoon
provides the best scenario for individuals seeking to in-
crease whole-body fat oxidation during aerobic exercise.
Fig. 4 Maximum oxygen uptake (VO
2max
) in absolute terms in the morning, and in the afternoon, following the ingestion of caffeine or the placebo.
Panel a: Individual observations for each subject (grey lines), and the mean for all subjects (black line). Panel b: Individual observations for each subject
(black dots), standard deviation and minimum/maximum values (box-and-whisker plot), and Pvalue obtained by two-way ANOVA. Similar letters (i.e. a-
a; b-b, etc) indicate significant post hoc differences. VO
2max
relative to weight in the morning, and in the afternoon, following the ingestion of caffeine
or the placebo. Panel c: Individual observations for each subject (grey lines), and the mean for all subjects (black line). Panel d: Individual observations
for each subject (black dots), standard deviation and minimum/maximum values (box-and-whisker plot), and the Pvalue obtained by two-way
ANOVA. Similar letters (i.e. a-a; b-b, etc.) indicate significant post hoc differences
Ramírez-Maldonado et al. Journal of the International Society of Sports Nutrition (2021) 18:5 Page 6 of 9

Diurnal variation of MFO, Fat
max
and VO
2max
The present findings provide further evidence regarding
the diurnal variation of MFO and Fat
max
, which have
been reported higher in the afternoon than in the morn-
ing [7–9]. It should be noted that these previous studies
were conducted using a treadmill graded exercise test to
measure these variables. In the present work, a cycloerg-
ometer graded exercise test was used. Thus, together,
these results suggest that the diurnal variations in MFO
and Fat
max
are independent of subject characteristics
and of the ergometer and protocol used to assess the
whole-body fat oxidation rate during exercise. A number
of studies have reported athletes to show better endur-
ance performance during the afternoon than the early
morning and late evening [10,35], a finding with which
the present results agree. However, in one study con-
ducted in trained male athletes, no differences in VO
2max
were seen between the morning and the afternoon [9].
With respect to this particular variable, the discrepancy
might be explained by the different ergometers used (i.e.,
a cycloergometer in the present work, and a treadmill in
the latter work), or the different biological characteristics
of the study subjects, or the different fasting times before
conducting the exercise test (3 h vs. 7–10 h respectively).
Endurance performance peaks in the afternoon usually
coinciding with the highest core body temperature
reached during the day [36]. This temperature increases
energy metabolism, improves muscle compliance, and fa-
cilitates actin-myosin cross bridging [11]. Moreover, the
exercise-induced catecholamine peak is higher in the
afternoon than in the morning [10,11]. This catechol-
amine release promotes an increase in lipolysis in both
skeletal muscle and adipose tissue [11,35], raising the
plasma fatty acid content and explaining the higher fat
oxidation rates observed in the afternoon. Since the
present work collected no data on body core temperature
or catecholamine release during exercise, further studies
will be needed if these variables are to be better linked to
the physiological mechanisms behind the observed diurnal
variation in VO
2max
, MFO and Fat
max
.
Caffeine ingestion, MFO, Fat
max
and VO
2max
The results of the current study support the use of caf-
feine as an ergogenic aid to raise fat oxidation during ex-
ercise, as well as to increase VO
2max
, and agree with the
findings of previous investigations showing that caffeine
improves fuel oxidation during prolonged exercise [20–
22] and enhances endurance performance [12]. The
present results also agree with those obtained by Gutiér-
rez-Hellín et al. [19] who reported 3 mg/kg caffeine to
increase MFO in healthy active young men, as well as
those reported by Dodd et al. [37] who indicate that 5
mg/kg of caffeine improved VO
2max
in naive caffeine
consumers. The higher MFO, Fat
max
and VO
2max
values
recorded in the present work after caffeine ingestion
may be explained by (i) an enhancement of fatty acid
mobilization and oxidation, aided by an increase in the
release of epinephrine, (ii) a blockage of the A
1
,A
2A
, and
A
2B
adenosine receptors, thus promoting the release of
acetylcholine and dopamine which dampens pain per-
ception, blunts perceived exertion, and delays fatigue
[16,38–40], (iii) an increase in motor unit recruitment,
which results in higher rates of muscular contraction
and alertness [16], and/or (iv) an increase in muscle oxy-
gen saturation that might facilitate the use of fat at mod-
erate exercise intensities and lead to higher VO
2
values
at maximal exercise intensity [18].
Effects of caffeine intake on the diurnal variation in MFO,
Fat
max
and VO
2
max
Mora-Rodríguez et al. [24] reported the acute ingestion of
caffeine (3 mg/kg) to reverse the morning reduction in
muscle performance - in fact to allow comparable muscle
performance to those seen in the afternoon. These find-
ings suggest that caffeine ingestion in the morning could
be used by athletes as an ergogenic aid to help them avoid
morning-induced reduction in muscle performance. In
addition, Boyett et al. [23], who investigated whether the
effect of caffeine on athletes’performance in a 3 km cyc-
ling time trial was influenced by the time of day and train-
ing status, concluded that caffeine enhanced cycling
performance more in the morning than in the evening.
These findings are partially in line with those of the
present study, suggesting that acute caffeine intake before
exercise serves as an effective ergogenic aid for reversing
morning-induced reductions in resistance exercise per-
formance and endurance-like performance.
The present study suffers from the limitation that
body temperature and blood variable data were not col-
lected during the graded protocol test, precluding any
confirmation that metabolic and hormonal variables play
a role in the diurnal variation of MFO, Fat
max
and/or
VO
2max
. Moreover, we did not control the sleep quality
and quantity of the participants. Further, the present
study was performed in active men; the results cannot,
therefore, be directly extrapolated to women or seden-
tary populations, etc. Finally, the sample size was rela-
tively small.
Practical applications
Caffeine intake increases MFO and Fat
max
as well as
VO
2
max independent of the time of day.
The highest values for these variables were all
obtained in the afternoon after caffeine intake.
Caffeine increases MFO in the morning to a value
similar to that seen without caffeine in the
afternoon.
Ramírez-Maldonado et al. Journal of the International Society of Sports Nutrition (2021) 18:5 Page 7 of 9

A combination of acute caffeine intake and exercise
at moderate intensity in the afternoon provides the
best scenario for individuals seeking to increase
MFO.
Conclusions
In summary, the acute ingestion of caffeine (3 mg/kg)
30 min prior to a graded exercise test increased the
MFO, Fat
max
and VO
2max
in active caffeine-naïve men
independent of the time of day. Further, the existence of
a diurnal variation in MFO, Fat
max
and VO
2max
was con-
firmed, with values for all being higher in the afternoon
than in the morning. The present findings also support
the notion that caffeine ingestion in the morning helps
to increase MFO and Fat
max
levels during exercise in the
afternoon. These results support the use of caffeine as
an ergogenic aid during training or competition during
the morning. The combination of acute caffeine intake
and exercise at moderate intensity in the afternoon
seems to be the best scenario for individuals seeking to
increase the amount of fat utilized during continuous
aerobic exercise. Whether higher doses of caffeine in-
duce greater effects on whole-body fat oxidation during
graded exercise tests and further improves endurance
performance remains to be investigated.
Abbreviations
VO
2max
:Maximal oxygen uptake; MFO: Maximal fat oxidation during a graded
exercise test; Fat
max
: Intensity of exercise that elicits maximal fat oxidation
during exercise
Acknowledgements
We are grateful to Adrian Burton for language and editing assistance and
to Harrison Sport Nutrition (HSN) store for its technical support.
Authors’contributions
MRM carried out the study procedures, and drafted the manuscript; LJF
conceived of the study, discussed the results, revised the manuscript and
approved the final version; JcC discussed the results, revised the manuscript
and approved the final version; JRR conceived of the study, discussed the
results, revised the manuscript and approved the final version; FAG
conceived of the study, and participated in its design and coordination,
drafted the manuscript and revised and approved the final version.
Funding
This study was supported by the University of Granada Plan Propio de
Investigación 2016—Excellence actions: Unit of Excellence on Exercise and
Health (UCEES) and the Junta de Andalucía, Consejería de Conocimiento,
Investigación y Universidades (ERDF: SOMM17/6107/UGR).
Availability of data and materials
The datasets used and/or analysed during the current study are available
from the corresponding author on reasonable request.
Ethics approval and consent to participate
All subjects provided oral and written informed consent before their enrolment.
Procedures were performed in accordance with the latest revised Declaration
of Helsinki (2013). The University of Granada Research Ethics Committee
approved the present project (N° 507/CEIH/2018).
Consent for publication
Not applicable.
Competing interests
The authors have no conflicts of interest to declare. The results of the study
are presented clearly, honestly, and without fabrication, falsification, or
inappropriate data manipulation.
Author details
1
Department of Physiology. Faculty of Medicine, University of Granada, Av.
Conocimiento S/n, 18011 Granada, Spain.
2
PROFITH “PROmoting FITness and
Health Through Physical Activity”Research Group, Department of Physical
Education and Sport, Faculty of Sport Sciences, University of Granada,
Granada, Spain.
3
Centre for Sport Studies, Rey Juan Carlos University, Madrid,
Spain.
Received: 13 July 2020 Accepted: 8 December 2020
References
1. Barnes KR, Kilding AE. Strategies to improve running economy. Sport Med.
2015;45:37–56 Available from: http://www.ncbi.nlm.nih.gov/
pubmed/25164465.
2. Fernández-Verdejo R, Bajpeyi S, Ravussin E, Galgani JE. Metabolic flexibility
to lipid availability during exercise is enhanced in individuals with high
insulin sensitivity. Am J Physiol Endocrinol Metab. 2018;315:E715–E722.
Available from: https://www.physiology.org/doi/https://doi.org/10.1152/
ajpendo.00126.2018.
3. Maunder E, Plews DJ, Kilding AE. Contextualising maximal fat oxidation during
exercise: determinants and normative values. Front Physiol. 2018;9:599.
4. Venables MC, Jeukendrup AE. Endurance training and obesity: effect on
substrate metabolism and insulin sensitivity. Med Sci Sports Exerc United
States. 2008;40:495–502.
5. Hearris M, Hammond K, Fell J, Morton J. Regulation of muscle glycogen
metabolism during exercise: implications for endurance performance and
training adaptations. Nutrients. 2018;10:298 Available from: http://www.
mdpi.com/2072-6643/10/3/298.
6. Souissi N, Bessot N, Chamari K, Gauthier A, Sesboüé B, Davenne D. Effect of
time of day on aerobic contribution to the 30-s Wingate test performance.
Chronobiol Int. 2007;24:739–48.
7. Darvakh H, Nikbakht M, Shakerian S, Sadat Mousavian A. Effect of Circadian
Rhythm on Peak of Maximal Fat Oxidation on Non-Athletic Men. Zahedan J
Res Med Sci. 2014;16:8–11.
8. Mohebbi H, Azizi M. Maximal fat oxidation at the different exercise intensity
in obese and normal weight men in the morning and evening. J Hum
Sport Exerc. 2011;6:49–58.
9. Amaro-Gahete FJ, Jurado-Fasoli L, Triviño AR, Sanchez-Delgado G, De-la-O
A, Helge JW, et al. Diurnal variation of maximal fat-oxidation rate in trained
male athletes. Int J Sports Physiol Perform. 2019;14:1140–6.
10. Drust B, Waterhouse J, Atkinson G, Edwards B, Reilly T. Circadian rhythms in
sports performance—an update. Chronobiol Int. 2005;22:21–44.
11. Teo W, Newton MJ, McGuigan MR. Circadian rhythms in exercise
performance: implications for hormonal and muscular adaptation. J Sport
Sci Med. 2011;10:600–6.
12. Grgic J, Mikulic P, Schoenfeld BJ, Bishop DJ, Pedisic Z. The influence of
caffeine supplementation on resistance exercise: a review. Sport Med. 2019;
49:17–30.
13. Southward K, Rutherfurd-Markwick KJ, Ali A. The effect of acute caffeine
ingestion on endurance performance: a systematic review and meta–
analysis. Sport Med 2018;48:1913–1928. Available from: http://link.springer.
com/https://doi.org/10.1007/s40279-018-0939-8.
14. Aguilar-Navarro M, Muñoz G, Salinero J, Muñoz-Guerra J, Fernández-Álvarez
M, Plata M, et al. Urine caffeine concentration in doping control samples
from 2004 to 2015. Nutrients. 2019;11:286 Available from: http://www.mdpi.
com/2072-6643/11/2/286.
15. Grgic J, Grgic I, Pickering C, Schoenfeld BJ, Bishop DJ, Pedisic Z. Wake up
and smell the coffee: caffeine supplementation and exercise
performance—an umbrella review of 21 published meta-analyses. Br J
Sports Med. 2019;bjsports-2018-100278. Available from: http://bjsm.bmj.
com/lookup/doi/https://doi.org/10.1136/bjsports-2018-100278.
16. Goldstein ER, Ziegenfuss T, Kalman D, Kreider R, Campbell B, Wilborn C,
et al. International society of sports nutrition position stand: caffeine and
performance. J Int Soc Sports Nutr. 2010;7:1–15.
Ramírez-Maldonado et al. Journal of the International Society of Sports Nutrition (2021) 18:5 Page 8 of 9

17. Burke LM, Hawley JA. Swifter, higher, stronger: What’s on the menu?
Science. 2018;362:781–787. Available from: https://www.sciencemag.org/
lookup/doi/https://doi.org/10.1126/science.aau2093.
18. Ruíz-Moreno C, Lara B, Brito de Souza D, Gutiérrez-Hellín J, Romero-
Moraleda B, Cuéllar-Rayo Á, et al. Acute caffeine intake increases muscle
oxygen saturation during a maximal incremental exercise test. Br J Clin
Pharmacol. 2019;bcp.1418.
19. Gutiérrez-Hellín J, Del Coso J. Effects of p-Synephrine and caffeine ingestion
on substrate oxidation during exercise. Med Sci Sport Exerc. 2018;50:1899–
906.
20. Schubert MM, Hall S, Leveritt M, Grant G, Sabapathy S, Desbrow B. Caffeine
consumption around an exercise bout: effects on energy expenditure,
energy intake, and exercise enjoyment. J Appl Physiol 2014;117:745–754.
Available from: https://www.physiology.org/doi/https://doi.org/10.1152/
japplphysiol.00570.2014.
21. Anderson DE, Hickey MS. Effects of caffeine on the metabolic and
catecholamine responses to exercise in 5 and 28 degrees C. Med Sci Sports
Exerc, Available from. 1994;26:453–8http://www.ncbi.nlm.nih.gov/pubmed/
8201901.
22. Cruz R, de Aguiar R, Turnes T, Guglielmo L, Beneke R, Caputo F. Caffeine
affects time to exhaustion and substrate oxidation during cycling at
maximal lactate steady state. Nutrients. 2015;7:5254–64 Available from:
http://www.mdpi.com/2072-6643/7/7/5219.
23. Boyett J, Giersch G, Womack C, Saunders M, Hughey C, Daley H, et al. Time
of Day and training status both impact the efficacy of caffeine for short
duration cycling performance. Nutrients. 2016;8:639. Available from: http://
www.mdpi.com/2072-6643/8/10/639.
24. Mora-Rodríguez R, Pallarés JG, López-Samanes Á, Ortega JF, Fernández-Elías
VE. Caffeine ingestion reverses the circadian rhythm effects on
neuromuscular performance in highly resistance-trained men. PLoS One.
2012;7.
25. Souissi Y, Souissi M, Chtourou H. Effects of caffeine ingestion on the diurnal
variation of cognitive and repeated high-intensity performances. Pharmacol
Biochem Behav, Available from. 2019;177:69–74 http://www.ncbi.nlm.nih.
gov/pubmed/30611752.
26. Souissi M, Chtourou H, Abedelmalek S, Ben GI, Sahnoun Z. The effects of
caffeine ingestion on the reaction time and short-term maximal performance
after 36h of sleep deprivation. Physiol Behav. 2014;131:1–6 Available from:
https://linkinghub.elsevier.com/retrieve/pii/S0031938414002030.
27. Horne JA, Ostberg O. A self-assessment questionnaire to determine
morningness-eveningness in human circadian rhythms. Int J Chronobiol.
1976;4:97–110.
28. Amaro-Gahete FJ, Sanchez-Delgado G, Jurado-Fasoli L, De-la-O A, Castillo
MJ, Helge JW, et al. Assessment of maximal fat oxidation during exercise: a
systematic review. Scand J Med Sci Sports. 2019;sms.13424. Available from:
https://onlinelibrary.wiley.com/doi/abs/https://doi.org/10.1111/sms.13424.
29. Frandsen J, Pistoljevic N, Quesada JP, Amaro-Gahete FJ, Ritz C, Larsen S,
et al. Menstrual cycle phase does not affect whole body peak fat oxidation
rate during a graded exercise test. J Appl Physiol. 2020;128:681–7.
30. Amaro-Gahete FJ, Sanchez-Delgado G, Alcantara JMA, Martinez-Tellez B,
Acosta FM, Helge JW, et al. Impact of data analysis methods for maximal fat
oxidation estimation during exercise in sedentary adults. Eur J Sport Sci.
2019;1–10. Available from: https://www.tandfonline.com/doi/full/https://doi.
org/10.1080/17461391.2019.1595160.
31. Frayn KN. Calculation of substrate oxidation rates in vivo from gaseous
exchange. J Appl Physiol. 1983;55:628–34 Available from: http://www.ncbi.
nlm.nih.gov/pubmed/6618956.
32. Amaro-Gahete FJ, Sanchez-Delgado G, Helge JW, Ruiz JR. Optimizing
maximal fat oxidation assessment by a treadmill-based graded exercise
protocol: when should the test end? Front Physiol. 2019;10:909. Available
from: https://www.frontiersin.org/article/https://doi.org/10.3389/fphys.2019.
00909/full.
33. Tanaka H, Monahan K, Seals D. Age-predicted maximal heart rate revisited. J
Am Coll Cardiol. 2001;37:153–6.
34. Beltz NM, Gibson AL, Janot JM, Kravitz L, Mermier CM, Dalleck LC. Graded exercise
testing protocols for the determination of VO 2 max: historical perspectives,
Progress, and future considerations. J Sports Med. 2016;2016:1–12.
35. Atkinson G, Todd C, Reilly T, Waterhouse J. Diurnal variation in cycling
performance: influence of warm-up. J Sports Sci. 2005;23:321–9.
36. Kim HK, Konishi M, Takahashi M, Tabata H, Endo N, Numao S, et al. Effects of
acute endurance exercise performed in the morning and evening on
inflammatory cytokine and metabolic hormone responses. PLoS One. 2015;
10:1–10.
37. Dodd SL, Brooks E, Powers SK, Tulley R. The effects of caffeine on graded
exercise performance in caffeine naive versus habituated subjects. Eur J
Appl Physiol Occup Physiol. 1991;62:424–9.
38. Doherty M, Smith PM. Effects of caffeine ingestion on rating of perceived
exertion during and after exercise: a meta-analysis. Scand J Med Sci Sport.
2005;15:69–78.
39. LeBlanc J, Jobin M, Cote J, Samson P, Labrie A. Enhanced metabolic
response to caffeine in exercise-trained human subjects. J Appl Physiol.
1985;59:832–7.
40. Ganio MS, Klau JF, Casa DJ, Armstrong LE, Maresh CM. Effect of caffeine on
sport-specific endurance performance: a systematic review. J Strength Cond
Res. 2009;23:315–24.
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Ramírez-Maldonado et al. Journal of the International Society of Sports Nutrition (2021) 18:5 Page 9 of 9