Subjects
Fifteen active men, aged 32 ± 7 years, volunteered to participate 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/m2, (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 including three or more training sessions/week [3.6 ± 0.2 sessions/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 Committee 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 experimental design involving a graded exercise test performed 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 Nutrition (HSN) Store, Granada, Spain]) or a 100% pure microcrystalline cellulose placebo [Acofarma, Madrid, 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 previous studies reporting caffeine to be effective at increasing 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 recipients; 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, Fatmax-morning, and VO2max-morning), and between 5 and 8 pm in the afternoon (providing MFO-afternoon, Fatmax-afternoon, and VO2max-afternoon). The order of (i) the time of the day when the exercise tests were performed, and (ii) the administration 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/m2). The body weight measured on this day was used in the dosage calculations for the entire 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 determine 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 before 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 motorized 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. Thereafter, 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 subsequent 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 before beginning a maximal graded exercise test to measure their VO2max. This began with the same warm-up protocol, followed by increments of 50 W every minute until self-reported exhaustion [29]. Indirect calorimetry data were registered using a CPX Ultima CardiO2 breath-by-breath gas analyzer (Medical Graphics Corp., St. Paul, MN, USA). A prevent™ metabolic flow sensor (Medgraphics) fitted to a model 7400 oronasal 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 exercise. The gas analyzer was calibrated immediately before each graded exercise, according to the manufacturer’s recommendations.
Submaximal graded exercise test
The VO2 and VCO2 data derived from the last 60 s of each graded exercise stage were taken into account [30]. Fat oxidation rates were estimated from the stoichiometric equation of Frayn, assuming urinary nitrogen excretion to be negligible [31]. MFO and Fatmax were determined by plotting fat oxidation values (dependent variable) against the relative exercise intensity (independent 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 VO2max to have been reached were: (i) attaining a steady (increase < 2 ml/kg/min) in VO2 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 respiratory exchange ratio of > 1.1 [34]. When these criteria were not met, peak oxygen consumption was taken into account (i.e., the highest VO2 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 minimum 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 analysis of variance (time-of-the day x substance) was used to compare MFO, Fatmax and VO2max under different study conditions. When a significant F value was obtained, 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 calculate the effect of the time of the day and substance provided on MFO, Fatmax and VO2max using pairwise tests. Significance was set at P < 0.05. Lastly, we also calculated the standardized effect sizes using Cohen’s d coefficients. Graphs were plotted using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA).