Habitual Caffeine Consumption and Training Status Affect the Ergogenicity of Acute Caffeine Intake on Exercise Performance

Abstract

Background:

Acute caffeine ingestion can improve exercise performance. Interplay between caffeine habituation and training status on the performance-enhancing effect of caffeine is unknown.

Hypothesis:

Habitual caffeine consumption and training status affect the ergogenicity of pre-exercise caffeine intake on exercise performance.

Study Design:

Double-blind, placebo-controlled, counterbalanced experimental design.

Level of Evidence:

Level 3.

Methods:

Eighty physically inactive men were randomized into 1 of 4 groups: caffeine supplementation (CAF), caffeine supplementation + exercise training (CAFEXE), placebo (PLA), and placebo + exercise training (PLAEXE); high-intensity interval training and caffeine were administered for 9 and 8 weeks, respectively. Data were collected pre-test, mid-test, post-test, and delayed post-test, each including 2 experiment sessions (3 mg/kg caffeine or placebo), with an additional experiment session post-test (6 mg/kg caffeine). In each experiment session, 45-min after consuming a placebo or caffeine, a 3-km running test and a Wingate power test were performed.

Results:

Pre-exercise ingestion of 3 mg/kg caffeine improved 3-km running time and mean power output (MPO) in all groups at all stages (P < 0.05); this effect was higher in trained than in untrained volunteers (P < 0.05). Habitual caffeine consumption reduced the ergogenic effect of caffeine in both aerobic and anaerobic trials (P < 0.05); 6 mg/kg caffeine enhanced this decrease only in CAFEXE (P < 0.05). Short-term caffeine withdrawal augmented the reduced ergogenic effect of caffeine on 3-km running performance and MPO in CAF and CAFEXE (P < 0.05).

Conclusion:

Habituation to caffeine and training status could partially influence the ergogenic effects of caffeine on exercise performance.

Clinical Relevance:

Regular caffeine consumption leads to some degree of tolerance and decreases its ergogenicity. A pre-exercise increase in caffeine dosage in trained people and short-term caffeine withdrawal in both trained and untrained people could compensate for the reduced caffeine ergogenicity in young men.

Caffeine (1,3,7-trimethylxanthine) is one of the most well-established ergogenic aids, with performance-enhancing effects across a wide range of exercise types, including strength,^27,28 power,^2,35 aerobic endurance,^2,14,35 and muscular endurance exercises.^27,51 Caffeine is consumed daily by 80% of the world’s population, ³⁰ and >70% of athletes use it before or during sports competitions. ¹⁸ Athletes of individual sports or athletes of sports with an aerobic-like nature are more prone to using caffeine in competitions. ¹ The ergogenic effects of caffeine on exercise performance appear to be related primarily to its binding to adenosine A₁ and A_2A receptors, increasing the release of norepinephrine and dopamine and reinforcing alertness and attention as well as attenuating perceived exertion, fatigue, and pain during exercise. ³⁸ Enhanced fat oxidation, ²⁹ increased muscle oxygen saturation, ⁴² and local changes within the exercising muscle, ⁴⁸ have also been suggested to explain the ergogenic effect of caffeine.

While habitual use of caffeine is frequently identified as a factor altering the acute exercise response to its supplementation, ³⁷ studies in this field illustrate conflicting results.^{6,7,14,17,19,26,27,35} For example, Grgic and Mikulic ²⁶ showed that habitual caffeine intake might not modify the ergogenic effects of acute caffeine supplementation on resistance exercise, jumping, and Wingate performance in resistance-trained male athletes. The same improvement in time to exhaustion, countermovement jump height, and rate of perceived exertion in both higher and lower caffeine-consuming professional male athletes following acute caffeine ingestion was reported by Apostolidis et al. ² On the other hand, Bell and McLellan ⁷ revealed that endurance exercise time to exhaustion differed between users and nonusers following acute caffeine intake, with the ergogenic effect being greater and lasting longer in nonusers. In addition, Lara et al ³⁵ demonstrated that, although caffeine remained ergogenic throughout 20 days, its ergogenic effects were reduced, suggesting that partial habituation may occur. Similar observations have also been reported by Beaumont et al ⁶ after 28 days of caffeine ingestion. Interestingly, it has been proposed that ingestion of a pre-exercise caffeine dose greater than that habitually consumed may compensate for the decrease in ergogenic effects. ⁴⁰ Short-term caffeine withdrawal before the competition has also been suggested in this regard. ²² However, these suggestions have been challenged by others.^31,52

In addition to caffeine habituation, the level of physical fitness or training status is considered another factor that can modify the acute ergogenic effects of caffeine on exercise performance. ²⁵ Again, the results are inconsistent in this regard.^4,9,16 For example, Collomp et al ¹⁶ reported that only trained athletes significantly improved their swimming velocity after caffeine ingestion. On the other hand, O’Rourke et al ³⁹ showed that both well-trained and recreational runners improved their outdoor 5-km time-trial performance following caffeine intake, with no between-group differences. Likewise, Astorino et al ⁴ reported similar improvements in endurance athletes and active men who completed a 10-km time trial after acute caffeine ingestion.

Consequently, there are mixed results, and the best procedure to optimize pre-exercise caffeine use remains unclear. Given that both habitual caffeine consumption and training status may affect the ergogenicity of the substance on exercise performance, and, to the best of our knowledge, whether the interplay between these factors affects the ergogenicity of acute caffeine ingestion on exercise performance has not been reported in the literature, this study aimed to answer the following questions: does habitual caffeine use affect its ergogenic effects? Can training status modify the ergogenic effects of acute caffeine intake on exercise performance? Does a pre-exercise caffeine dose in excess of its habitual intake help to compensate for the possible reduction in its ergogenic effects? Might a probable reduction in caffeine ergogenicity be offset by a short-term caffeine withdrawal before exercise?

Methods

Study Design

A double-blind, placebo-controlled, and counterbalanced experimental design was used in this study (Figure 1). A researcher who was not involved in data collection and analysis coordinated the study procedures. The participants completed a familiarization session after a caffeine-free week. During this session, volunteers were familiarized with the laboratory environment, testing procedures, and exercise training protocol after being medically screened. Then, body mass and height were measured. To ensure that the learning effect was minimal, they also performed a 10-minute treadmill exercise (walking, 5 minutes, 4 km/h; and running, 5 minutes, 7 km/h, at an incline of 0%) followed by 5 minutes cycling on an ergometer cycle at a workload of 150 W. Next, participants completed a maximal aerobic exercise test to evaluate maximal oxygen consumption (VO₂max) at least 48 hours after the familiarization session. To minimize possible group differences, participants were matched according to age, body mass, and VO₂max and randomized on a 1:1:1:1 basis to 1 of 4 groups (20 subjects in each group): caffeine supplementation (CAF), caffeine supplementation + exercise training (CAFEXE), placebo (PLA), and placebo + exercise training (PLAEXE).

Figure 1.

Schematic of (a) study design and (b) experimental protocol. CAF, caffeine supplementation; CAFEXE, caffeine supplementation + exercise training; PLA, placebo; PLAEXE, placebo + exercise training; VO₂max, maximal oxygen consumption; HIT, high intensity interval training.

With at least a 5-day interval after the VO₂max determination session, data collection continued in 4 stages: pre-test, mid-test, post-test, and delayed post-test. Each consisted of 2 separate experimental sessions with a time interval of 72 hours, except the post-test, which consisted of 3 separated experiment sessions. On the experiment days, the volunteers consumed a cup of lightly brewed green tea (containing ~15 mg of caffeine) at 6:00 a.m. (after 7-8 hours of overnight fasting) followed by breakfast at 8:00 a.m., both of which took place under the supervision of the researchers in the student residence. This titrated approach minimized the negative effects of acute caffeine withdrawal and the reversal of withdrawal effects on exercise performance.

At the pre-test, mid-test, and delayed post-test, the participants underwent 2 experiment sessions under different conditions: 3 mg/kg of caffeine (Caffeine, Olimp Laboratories) or placebo (250 mg cellulose). At the post-test, there was a third experimental session in which participants received 6 mg/kg of caffeine (post-test-6). The order of the sessions was random and counterbalanced. Participants attended the experiment site at 11:00 a.m. and immediately ingested the substances in opaque capsules with a glass of water (approximately 3 hours after breakfast to maintain the same absorption time of the substance and 45 minutes before the onset of the trial to allow for caffeine absorption as peak plasma caffeine concentration is obtained 15 and 120 minutes after oral ingestion).^20,36 During the break between caffeine ingestion and the onset of the trial, the participants rested in a lying position and were allowed to drink only plain water. Participants then completed an aerobic endurance performance test followed by an anaerobic power test with a 20-minute rest between assessments. It was shown that the sequence of aerobic endurance performance test before an anaerobic power test allowed both tests to be administered during the same session without compromising aerobic endurance and anaerobic power. ⁴⁷

Except for the pre-test stage, the volunteers in the next 7 experiment sessions, depending on the pre-exercise substance consumption and the grouping, received a second capsule containing either caffeine or a placebo immediately after completing the test (ie, if a subject from the CAF groups consumed caffeine at first, he received a second capsule containing placebo, and vice versa. The second capsule for PLA subjects as well as for all subjects at the delayed post-test filled with cellulose). All participants completed the described procedure, which was repeated carefully on subsequent experimental days. At 48 hours after the last pre-test session, the training program was started and continued for 4 weeks. Next, the mid-test was performed 72 hours after the last training session. The second 4-week training program started 48 hours after the last mid-test experiment. The post-test was then performed 72 hours after the last training session. After a caffeine-free week to wash out substances, participants performed the delayed post-test experiments 72 hours after the last training session. During the administration of the high-intensity interval training (HIT) program, the nonexercising participants watched the training in a sitting position. The subjects’ diet was the same 24 hours before all the experiments. Verbal encouragement to give maximum effort for each trial was standardized and provided throughout each experiment and training session. Furthermore, no visual feedback from the performances was provided, with the exception of elapsed distance in the time trial. The indoor trials were conducted in a laboratory with a dry temperature of 21.1 ± 0.4 °C and a relative humidity of 30 ± 10%. Except for the VO₂max test sessions conducted from 9:00 a.m. to 14:00 p.m., all other experimental trials and training sessions were conducted at the same time of the day (11:00 a.m.-13:00 p.m.) to avoid the effect of circadian variation on the results. All procedures were undertaken according to the ethical standards of the latest version of the Declaration of Helsinki and approved by the Ethics Committee of Istanbul Esenyurt University with the number 2023/09-10, where the study was conducted. The study was registered with the Iranian Registry of Clinical Trials (IRCT20231004059615N1).

Participants

An a priori power analysis (G*Power Version 3.1.9.4) indicated that the minimum required sample size was 68 participants. This calculation was based on a small effect size (ES) ƒ of 0.15, an alpha error of 0.05, a statistical power of 90%, and a correlation value of 0.50 for the repeated measures, within-between analysis of variance (ANOVA) design with 4 groups. Accordingly, 80 healthy and physically inactive men (age, 20.7 ± 1.9 years; height, 176 ± 8.2 cm; body mass, 80.08 ± 7.6 kg; VO₂max, 40.1 ± 4.7 ml/kg/min) participated in this study after being aware of the exact procedures, including any risks and benefits, and completing written informed consent. The inclusion criteria were as follows: (1) nonusers of caffeine (naïve consumer, daily caffeine intake <25 mg/day, ²¹ (2) no sign of caffeine withdrawal effects (ie, headache, fatigue, decreased energy/activeness, decreased alertness, drowsiness, decreased contentedness, depressed mood, difficulty concentrating, irritability, and yawning) during a primary caffeine-free week, ³⁴ (3) free from neuromuscular and musculoskeletal disorders, (4) no participation in exercise training at least 6 months before enrollment in this study, (5) nonsmoker and nonalcoholic, and (f) not taking any medicine or supplement for at least 3 months before the study. The study volunteers were college students residing in the student residence who received the same diet, which was controlled by a nutritionist. However, participants were asked to record their food and drink intake using “MyFitnessPal” software during the study. Daily caffeine consumption was assessed using an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. ¹¹ Participants also received a list of caffeine-containing products that were prohibited from consumption during the study. In addition, volunteers were encouraged to maintain their habitual bedtime/wake-up schedule. Participants were excluded from the study if they met any of the following criteria: suffered from any pathology or injury; used any medications, supplements, or ergogenic aids that could potentially affect the study results; had a caffeine consumption >25 mg/day in their diet for >3 days during the study; or missed >5% of the training trials.

Maximal Aerobic Exercise Test

Approximately 3 hours after breakfast, all participants performed a progressive incremental test on the treadmill (Varilant) using the Bruce protocol to determine VO₂max. This protocol requires the participants to run for as long as possible on a treadmill, with speed and slope increased at timed intervals. To complete the Bruce test, the subjects warmed up for 10 minutes. The treadmill speed was then adjusted to 2.7 km/h and a grade of 10%. Every 3 minutes, the speed and grade of the treadmill were increased. The test was continued until the participant reached volitional exhaustion. Ventilation (VE), oxygen uptake (VO₂) and carbon dioxide output (VCO₂) were measured at 10-second intervals using online computer-assisted circuit spirometry (Ganshorn Medizin Electronic GmbH Power Cube-Ergo), which was calibrated before each test according to the manufacturer’s instructions. The highest 10-second VO₂ value during the incremental test was recorded as the VO₂max value if it coincided with at least 3 of the following criteria: (1) a plateau in VO₂ despite an increase in running speed; (2) a respiratory exchange ratio (RER) >1.20; (3) peak heartrate ≥ 90% of the age-predicted maximum; and/or (4) visible subject exhaustion. ⁴⁵

Aerobic Endurance Performance Test

The 3-km running time-trial performance was conducted on an outdoor track. Participants were given a warm-up, which included 5 minutes of jogging and 5 minutes of stretching and running. The test began after a 5-minute rest. Verbal encouragement was provided during the test. The test was performed on sunny days with an ambient temperature of 20 °C to 25 °C with 50% to 60% relative humidity, and a light breeze. ⁴⁵

Anaerobic Power Test

The 30-second Wingate Anaerobic Power Test, which assesses both the ATP-PC and glycolytic energy systems, ⁴⁶ is used widely as a valid and reliable predictor of anaerobic performance. ¹³ Therefore, participants performed the Wingate test on a mechanically braked cycle ergometer (Lode B.V. Medical Technology) against a resistance equivalent to 75 g/kg body mass. Before the test, volunteers completed a 5-minute standardized warm-up. Participants were instructed to begin pedaling, as fast as possible, against the inertial resistance of the ergometer, and then the appropriate load was applied. Participants were encouraged verbally to maintain maximal speed throughout the 30-second test. Mean power output (MPO) is defined as the average work output for the 30-second test period.

Exercise Training Protocol

A standardized warm-up was performed, including 5 minutes of jogging, followed by 5 minutes of supervised dynamic exercises (walking lunges, heel flicks, high knees, leg swings, and jumping jacks). Then, participants completed 6 to 10 bouts of 30-second all-out sprints separated by a 4-minute active recovery, followed by a 5-minute cool down. Participants trained 5 sessions per week for 4 consecutive weeks. Following the mid-test, the training program was continued for 4 more weeks. There was also an additional training week before the delayed post-test that was performed at exactly the same volume and intensity as week 8. The training programs involved a periodized increase in volume with a taper in the fourth and eighth weeks. The 9-week exercise training program is shown in Figure 2.

Figure 2.

Representation of the periodized exercise training program.

Caffeine Supplementation

Caffeine supplementation was started from the second session of the exercise training program and continued until the last post-test experiment. The participants in the caffeine groups received 3 mg/kg/day of caffeine under the same conditions as the experiments, at 11:00 a.m. and approximately 3 hours after breakfast. On training days, the exercise program started after a 45-minute rest in the lying position following caffeine ingestion. In the ninth week of training, caffeine supplementation was stopped, and participants received a placebo instead. Participants in the placebo groups consumed a capsule with the same appearance but filled with cellulose, which was ingested for the same duration and frequency as in the caffeine treatment.

Evaluation of Blinding

After each experiment session, the effectiveness of blinding was examined by asking participants the following question: “Which supplement do you think you have ingested?.” ⁴⁴ This question had 3 possible responses: (1) “caffeine,” (2) “placebo,” and (3) “I do not know.”

Statistical Analysis

The Shapiro-Wilk, Levene and Mauchly’s tests were used to verify the normality, homogeneity, and sphericity of the sample data variance. If the latter assumption presented a probability of P < 0.05, the Greenhouse-Geisser correction was used. A 2-way (group × time) repeated-measures analysis of variance (ANOVA) was conducted on the absolute changes (caffeine minus placebo condition values at each stage) in aerobic and anaerobic performances to test for possible differences across the times between the groups. In the case of a significant main effect, pairwise comparisons were conducted using the least significant difference multiple comparisons test. The ES was also calculated using the Hedge’s g and interpreted using thresholds of <0.20, 0.20 to 0.49, 0.50 to 0.79, and ≥0.8 for trivial, small, moderate, and large effects, respectively. ¹⁵ Statistical significance was set at P < 0.05. All statistical analyses were performed using the statistical package SPSS Version 27 and presented as means ± standard deviations.

Results

Of the 80 participants who entered the study, 73 met the study criteria and were included in the analysis (CAFEXE, n = 18; PLAEXE, n = 18; CAF, n = 19; PLA, n = 18). The mean scores of the groups in experimental trials are presented in Table 1.

Table 1.

Average scores of the study groups in experiment trials

	Pre-test		Mid-test		Post-test			Delayed post-test
	Placebo	Caffeine	Placebo	Caffeine	Placebo	Caffeine(3)	Caffeine(6)	Placebo	Caffeine
3-km running time (s)
PLAEXE	951 ± 44	934 ± 49	855 ± 41	832 ± 43	798 ± 43	775 ± 48	774 ± 40	798 ± 43	773 ± 40
CAFEXE	959 ± 35	941 ± 35	850 ± 37	832 ± 36	805 ± 39	787 ± 39	781 ± 41	806 ± 37	782 ± 38
CAF	947 ± 33	930 ± 34	944 ± 35	931 ± 37	944 ± 45	932 ± 47	932 ± 45	946 ± 41	928 ± 40
PLA	959 ± 41	942 ± 43	959 ± 44	941 ± 46	952 ± 51	935 ± 52	934 ± 53	950 ± 50	932 ± 49
MPO (w)
CAFEXE	525 ± 51	535 ± 53	548 ± 49	572 ± 50	577 ± 52	602 ± 53	601 ± 52	578 ± 50	602 ± 56
PLAEXE	537 ± 55	546 ± 56	566 ± 54	580 ± 57	582 ± 58	597 ± 58	604 ± 58	579 ± 57	600 ± 59
CAF	525 ± 48	535 ± 48	527 ± 49	533 ± 50	529 ± 49	534 ± 50	534 ± 49	531 ± 48	542 ± 48
PLA	518 ± 51	529 ± 52	519 ± 53	531 ± 54	521 ± 51	534 ± 53	533 ± 51	519 ± 53	530 ± 52

CAF, caffeine supplementation; Caffeine(3), ingesting 3 mg/kg of caffeine before exercise; Caffeine(6), ingesting 6 mg/kg of caffeine before exercise; CAFEXE, caffeine supplementation + exercise training; MPO, mean power output; PLA, placebo; PLAEXE, placebo + exercise training.

All data are presented as mean ± SD.

Assessment of Blinding

The blinding assessment answers were as follows: at the pre-test stage, in the first (true, 27; false, 30; unable, 16) and second (true, 27; false, 23; unable, 23) experimental sessions; at the mid-test stage, in the first (true, 24; false, 31; unable, 18) and second (true, 31; false, 25; unable, 17) experimental sessions; at the post-test stage, in the first (true, 20; false, 28; unable, 25), second (true, 21; false, 19; unable, 33), and third (true, 32; false, 25; unable, 16) experimental sessions; and at the delayed post-test stage, in the first (true, 18; false, 27; unable, 28) and second (true, 25; false, 31; unable, 17) experimental sessions.

Aerobic Endurance Performance

As the statistical analysis was conducted on absolute changes (caffeine minus placebo condition at each stage), higher absolute changes indicate a more favorable responsiveness to caffeine and improved performance. Conversely, the lower absolute changes indicate poor ergogenicity of the substance. The 2-way repeated-measures ANOVA showed significant main effects of group (F = 5.741; P < 0.01) and time (F = 4.019; P < 0.01) as well as a significant interaction of group × time (F = 3.174; P < 0.01) on absolute changes in aerobic endurance performance. Pre-exercise ingestion of 3 mg/kg caffeine improved 3-km running time in all groups at all stages (P < 0.05, moderate to large ESs). The pairwise comparisons for the ergogenicity of caffeine revealed no significant between-group differences in the pre-test (P > 0.05, trivial ESs;). At the mid-test, the PLAEXE group had significantly higher responsiveness to caffeine than the PLA (P = 0.04, ES = 0.6), CAF (P < 0.01, ES = 1.3), and CAFEXE (P = 0.04, ES = 0.6) groups. Significantly lower ergogenicity of caffeine was also observed in the CAF group than in the PLA (P < 0.04, ES = 0.7) and CAFEXE (P < 0.04, ES = 0.6) groups. The responsiveness to caffeine between CAFEXE and PLA was similar (P = 0.96, ES = 0.01). Similar results were obtained at the post-test-(3) (PLAEXE vs PLA: P = 0.03, ES = 0.7; PLAEXE vs CAF: P < 0.01, ES = 1.4; PLAEXE vs CAFEXE: P = 0.04, ES = 0.6; CAF vs PLA: P = 0.04, ES = 0.7; CAF vs CAFEXE: P = 0.03, ES = 0.7; CAFEXE vs PLA: P = 0.87, ES = 0.05). At the post-test-(6), the CAF group exhibited significantly lower ergogenicity of caffeine than the PLA (P = 0.03, ES = 0.78), CAFEXE (P < 0.01, ES = 1.3), and PLAEXE (P < 0.01, ES = 1.6) groups. In addition, the PLAEXE (P = 0.03, ES = 0.71) and CAFEXE (P = 0.04, ES = 0.61) groups had significantly higher responsiveness to caffeine than the PLA group. The responsiveness to caffeine was similar between CAFEXE and PLAEXE (P = 0.95, ES = 0.02). At the delayed post-test, the PLAEXE and CAFEXE groups revealed higher ergogenicity of caffeine compared with the PLA (P = 0.02, ES = 0.84; P = 0.03, ES = 0.67, respectively) and CAF (P = 0.02, ES = 0.8; P = 0.03, ES = 0.65, respectively) groups. Similar caffeine responsiveness scores were observed between CAFEXE and PLAEXE (P = 0.85, ES = 0.06) and between CAF and PLA (P = 0.96, ES = 0.01).

Post hoc comparisons for within-subjects effects on the absolute difference between caffeine and placebo conditions for the 3-km running time showed that the CAF group had significantly lower responsiveness to caffeine at mid-test, post-test-(3), and post-test-(6) compared with pre-test (P < 0.01, ES = 0.84; P = 0.02, ES = 0.54; P = 0.02, ES = 0.54, respectively) and delayed post-test (P < 0.01, ES = 0.83; P < 0.01, ES = 0.68; P < 0.01, ES = 0.7, respectively). The ergogenicity of caffeine between mid-test and post-test-(3) (P = 0.62, ES = 0.11), mid-test and post-test-(6) (P = 0.49, ES = 0.16), post-test-(3) and post-test-(6) (P = 0.87, ES = 0.03), and between pre-test and delayed post-test (P = 0.69, ES = 0.09) was not significant in the CAF group. In addition, the CAFEXE group presented a higher responsiveness to caffeine at post-test-(6) and delayed post-test compared with pre-test (P < 0.01, ES = 0.7; P < 0.01, ES = 0.82, respectively), mid-test (P < 0.01, ES = 0.71; P < 0.01, ES = 0.73, respectively), and post-test-(3) (P < 0.01, ES = 0.79; P < 0.01, ES = 0.74, respectively). There was no significant difference between pre-test and mid-test (P = 0.87, ES = 0.04), pre-test and post-test-(3) (P = 0.77, ES = 0.07), mid-test and post-test-(3) (P = 0.89, ES = 0.03), and post-test-(6) and delayed post-test (P = 0.9, ES = 0.03) for the ergogenicity of caffeine in the CAFEXE group. Moreover, higher ergogenicity of caffeine was observed in the PLAEXE group at mid-test (P = 0.03, ES = 0.53), post-test-(3) (P = 0.08, ES = 0.7), post-test-(6) (P = 0.04, ES = 0.5), and delayed post-test (P = 0.03, ES = 0.54) than pre-test. The ergogenicity of caffeine between mid-test and post-test-(3) (P = 0.97, ES = 0.00), mid-test and post-test-(6) (P = 0.87 ES = 0.04), mid-test and delayed post-test (P = 0.616, ES = 0.12), post-test-(3) and post-test-(6) (P = 0.88, ES = 0.03), post-test-(3) and delayed post-test (P = 0.65, ES = 0.1), and post-test-(6) and delayed post-test (P = 0.68, ES = 0.09) were not significant for the PLAEXE group. The caffeine ergogenicity in the PLA group was not significantly different across the times (P < 0.05, trivial ESs) (Figure 3).

Figure 3.

The absolute difference between caffeine and placebo conditions in aerobic endurance performance. Higher absolute changes indicate a more favorable responsiveness to caffeine and lower absolute changes indicate poor ergogenicity of the substance. Data are presented as mean ± SD. CAF, caffeine supplementation; CAFEXE, caffeine supplementation + exercise training; PLA, placebo; PLAEXE, placebo + exercise training.

^aP < 0.05 different from the corresponding pre-test.

^bP < 0.05 different from the PLA group.

^cP < 0.05 different from the PLAEXE group.

^dP < 0.05 different from the CAFEXE group.

Anaerobic Power Performance

Significant main effects of group (F = 6.648, P < 0.01), time (F = 12.151, P < 0.01), and the interaction of group × time (F = 9.252, P < 0.01) were observed on absolute changes in MPO by 2-way repeated-measures ANOVA. Pre-exercise ingestion of 3 mg/kg caffeine improved 3-km running time in all groups at all stages (P < 0.05, moderate to large ESs). There was no significant difference in the caffeine ergogenicity between groups in the pre-test stage (P > 0.05, trivial ESs). In comparison with the PLA (P < 0.01, ES = 1.27), CAFEXE (P < 0.01, ES = 0.96), and CAF (P < 0.01, ES = 2) groups, the PLAEXE group had significantly higher responsiveness to caffeine at the mid-test. The ergogenicity of caffeine in the CAF group was also significantly lower than that in PLA (P = 0.04, ES = 0.68) and CAFEXE (P = 0.01, ES = 0.8). The ergogenicity of caffeine on MPO was not significantly different between the CAFEXE and PLA groups (P = 0.54, ES = 0.18). In the post-test (3), similar results were obtained by the groups (PLAEXE vs PLA: P < 0.01, ES = 1.1; PLAEXE vs CAF: P < 0.01, ES = 1.8; PLAEXE vs CAFEXE: P < 0.01, ES = 0.9; CAF vs PLA: P = 0.03, ES = 0.67; CAF vs CAFEXE: P = 0.01, ES = 0.77; CAFEXE vs PLA: P = 0.67, ES = 0.12). At the post-test (6), significantly higher ergogenicity of caffeine was observed by PLAEXE and CAFEXE than PLA (P < 0.01, ES = 1.2; P < 0.01, ES = 0.87, respectively) and CAF (P < 0.01, ES = 1.88; P < 0.01, ES = 1.5, respectively). The CAF group also showed a significantly lower responsiveness to caffeine than the PLA group (P = 0.04, ES = 0.69). The responsiveness to caffeine between the CAFEXE and PLAEXE groups was similar (P = 0.5, ES = 0.2). PLAEXE and CAFEXE groups exhibited significantly higher responsiveness to caffeine than the PLA (P < 0.01, ES = 1; P < 0.0, ES = 0.97, respectively) and CAF (P < 0.01, ES = 0.98; P < 0.01, ES = 0.91, respectively) groups at the delayed post-test. The ergogenicity of caffeine on MPO was similar between the PLAEXE and CAFEXE groups (P = 0.57, ES = 0.18) and between the CAF and PLA groups (P = 0.96, ES = 0.01).

Post hoc comparisons for within-subjects effects on the absolute changes for the MPO revealed that the CAF group experienced significantly lower caffeine ergogenicity at mid-test, post-test-(3), and post-test-(6) compared with pre-test (P < 0.01, ES = 0.84; P < 0.01, ES = 0.76; P < 0.01, ES = 0.75, respectively) and delayed post-test (P < 0.01, ES = 1; P < 0.01, ES = 0.89; P < 0.01, ES = 0.91, respectively). The ergogenicity of caffeine between the mid-test, post-test-(3), and post-test-(6) was not significant for the CAF group (P > 0.05, trivial ESs). The CAFEXE group showed significantly higher responsiveness to caffeine at mid-test (P = 0.02, ES = 0.56), post-test (3) (P = 0.03, ES = 0.55), post-test (6) (P < 0.01, ES = 1.4) and delayed post-test (P < 0.01, ES = 0.88) compared with the pre-test values. The caffeine ergogenicity in post-test (6) and delayed post-test were also higher than in the mid-test (P < 0.01, ES = 0.78; P = 0.03, ES = 0.52, respectively) and post-test (3) (P < 0.01, ES = 0.83, P = 0.03, ES = 0.54, respectively). There were no significant differences between the mid-test and post-test (3) (P = 0.82, ES = 0.05) or between the post-test (6) and delayed post-test (P = 0.58, ES = 0.13). In addition, the PLAEXE group experienced significantly higher ergogenicity of caffeine at the mid-test (P < 0.01, ES = 1.34), post-test (3) (P < 0.01, ES = 1.26), post-test (6) (P < 0.01, ES = 1.25), and delayed post-test (P < 0.01, ES = 0.88) compared with the pre-test. The caffeine ergogenicity in the PLAEXE group was not significantly different between the mid-test, post-test-(3), post-test-(6), and delayed post-test (P > 0.05, trivial to small ESs). The ergogenicity of caffeine in the PLA group was not significantly different across the times (P > 0.05, trivial to small ESs) (Figure 4).

Figure 4.

The absolute difference between caffeine and placebo conditions for MPO. Higher absolute changes indicate a more favorable responsiveness to caffeine and lower absolute changes indicate poor ergogenicity of the substance. Data are presented as mean ± SD. CAF, caffeine supplementation; CAFEXE, caffeine supplementation + exercise training; MPO, mean power output; PLA, placebo; PLAEXE, placebo + exercise training.

^aP < 0.05 different from the corresponding pre-test.

^bP < 0.05 different from the PLA group.

^cP < 0.05 different from the PLAEXE group.

^dP < 0.05 different from the CAFEXE group.

Discussion

The primary aim of the present study was to investigate the effect of habitual caffeine intake and its interplay with training status on aerobic and anaerobic performance following acute caffeine ingestion. The secondary aim was to examine the effect of using a pre-exercise caffeine dose in excess of the volunteers’ habitual intake as well as short-term caffeine withdrawal to offset the reduction of its ergogenic effects in regular caffeine users. The HIT protocol was used as the training program because it has been shown to improve both aerobic and anaerobic capacities simultaneously. ¹⁰ Pre-exercise ingestion of 3 mg/kg caffeine improved 3-km running performance and MPO in all groups at all stages; this effect was more prominent in the trained subjects than in the corresponding untrained subjects. Regardless of training status, habitual caffeine consumption resulted in a certain level of tolerance that reduced, but did not entirely eliminate, the ergogenic effect of acute caffeine ingestion in both aerobic and anaerobic trials. Administration of 6 mg/kg caffeine compensated for the decrease in the ergogenic effect of caffeine on exercise performance in trained but not in untrained volunteers. In addition, short-term caffeine withdrawal magnified the reduced ergogenic effect of caffeine on 3-km running time-trial performance and MPO in both trained and untrained habitual caffeine-consuming participants.

Caffeine has been shown to elicit a variety of biological effects and has been gaining acceptance as an ergogenic aid that may mediate performance enhancement across a wide range of exercise types.^2,27,35 However, the ergogenicity of pre-exercise acute caffeine ingestion has become a challenge in habitual consumers, and studies in this regard have provided conflicting evidence.^{6,7,14,17,19,26} The current study demonstrates that when a moderate dose of caffeine is consumed daily, tolerance to this substance develops, regardless of training status. However, it still has the capacity to improve exercise performance after approximately 70 days of consecutive ingestion. These novel findings support and extend previous reports regarding the development of caffeine tolerance in habitual users,^6,7,19,35 and suggest that tolerance occurs during the first month of consumption and that no further tolerance to the ergogenic effects of caffeine will arise thereafter. In contrast to these findings, some studies have shown that pre-exercise caffeine ingestion leads to similar responses in caffeine users and nonusers.^14,17,26,43 One reason for this discrepancy may be the quantification of habitual caffeine intake. Using a Food Frequency Questionnaire is a well-known method of monitoring caffeine intake. However, it is possible that the habitual caffeine intakes of the participants in the studies are not entirely accurate because of the large variability in the caffeine content of commonly consumed products. ³ In addition to the Food Frequency Questionnaire, volunteers with no withdrawal symptoms during the initial 1-week free-caffeine period were included in this study. Their fasting serum caffeine concentration was also 0.00 to 0.07 μg/ml (data not shown). Therefore, naïve volunteers were recruited and habituation to caffeine was performed under the control of the researchers. Furthermore, the experimental protocol used in most studies involves a >12-hour abstinence period from caffeine,^14,17,26,43 which could have influenced the results because of the negative effects of acute caffeine withdrawal and/or the reversal of withdrawal effects.^3,32 In the current study, all participants consumed a cup of lightly brewed green tea approximately 6 hours before the exercise tests. The mean serum caffeine concentration at the beginning of the experimental sessions, immediately before the caffeine/placebo administration, was 0.07 ± 0.06 (0.02-0.17) μg/ml (data not shown). Age, sex, and genetic differences could also be considered other factors possibly involved in different results between studies on caffeine tolerance.

Interestingly, some studies that pointed to the lack of caffeine tolerance used acute doses higher than the participants’ usual caffeine consumption.^17,26 For example, Grgic and Mikulic ²⁶ stated that the ergogenic effects of 3 mg/kg caffeine on resistance exercise, jumping, and anaerobic powers may be because the dosage was equal to or greater than the amount of caffeine taken habitually for 21 out of the 24 resistance-trained males. On the other hand, studies that administered the same caffeine doses in both acute and chronic consumption reported less ergogenic effects following acute pre-exercise caffeine ingestion.^6,35 Accordingly, it has been suggested that any reductions in the ergogenic effect of caffeine because of its habitual use may be offset by the ingestion of a pre-exercise dosage greater than that habitually consumed.^37,40 To investigate this hypothesis, we used a dose of 6 mg/kg of caffeine (2-fold of daily intake by the volunteers) because previous studies have shown that caffeine benefits exercise performance only when consumed up to 6 mg/kg but not >6 mg/kg. ²⁴ The results revealed that a dose of 6 mg/kg of caffeine could largely compensate for its reduced ergogenic effect on time-trial endurance performance and MPO only in the CAFEXE group. Such an effect was observed neither in the subjects of the CAF group nor the placebo-consuming groups. In this context, Polito et al ⁴¹ reported that the use of 3 mg/kg doses of caffeine provides the same improvements in muscle strength as the administration of 6 mg/kg doses in resistance exercise-trained men who were low-habitual caffeine consumers (83.4 ± 9.2 mg/day). It appears that naïve and low-habitual caffeine consumers do not develop tolerance so, when consuming caffeine, they typically experience the physiological effects of the compound at lower levels of ingestion than habitual users, ⁴⁹ and additional consumption of the substance does not provide superiority. Overall, the efficiency of a pre-exercise increased caffeine dosage seems to be influenced by both habitual consumption and training status.

This study demonstrates that the short-term period of caffeine withdrawal could compensate for the decrease in ergogenic effects caused by its regular consumption in trained and untrained participants. Fisher et al ²² reported that caffeine withdrawal for 4 days increased physiological responses during 1-hour of running at 75% of VO₂max in caffeine-habituated (>600 mg/day) subjects. However, Van Soeren and Graham ⁵⁰ showed that 6 mg/kg caffeine improved time-to-exhaustion cycling trials in habitual caffeine users (mean daily caffeine intake 761 mg/day), irrespective of whether a 0-, 2-, and 4-day caffeine withdrawal period was imposed on habitual caffeine users. Irwin et al ³¹ also found that 3 mg/kg caffeine improved time-trial performance in trained cyclists with an average habitual caffeine intake of 240 mg/day, and the 4-day withdrawal period did not enhance caffeine ergogenicity. The duration of caffeine withdrawal may explain the discrepancy in results between our study and previous studies. The duration of caffeine withdrawal in the present study was 11 days, but a 4-day withdrawal period was conducted in the previous studies.^31,50 It was reported that the onset of symptoms of caffeine withdrawal starts 12 to 24 hours after caffeine cessation, peaks at 20 to 51 hours, and may last up to 9 days. ³⁴ Another possible reason for the difference in the findings could be related to interindividual variation (ie, caffeine tolerance may not occur in all people; therefore, the subsequent caffeine withdrawal period will be meaningless). In the present study, 5 out of 19 participants in the CAF group showed similar exercise performances over the 4 stages.

It has been concluded that short-term caffeine withdrawal before training/competition is associated with numerous adverse outcomes in habitual consumers, and such acute withdrawal symptoms close to the key competition can impair the quality of the training and may negatively affect athletes’ confidence and wellbeing and should be avoided.^12,40 In this study, 9 of 37 caffeine recipients showed mild caffeine withdrawal symptoms up to 72 hours after abstinence. All participants in the CAFEXE group completed their training program during the caffeine-free week; they experienced a nonsignificant increase with a small ES (P = 0.22, ES = 0.34) in perceived exertion during the first training session compared with the EXE group (data not shown). Furthermore, caffeine-habituated participants did not exhibit withdrawal symptoms during the experimental sessions. It appears that consuming a very small amount of caffeine (approximately 15 mg) in the form of green tea could probably prevent withdrawal symptoms and subsequent exercise performance impairment. Therefore, although caffeine abstinence in athletes who are habitual consumers can be associated with some side effects, reducing the dose of caffeine during the tapering period and increasing the dosage before the competition could be considered a suggestion that should be tested in future studies.

In the present study, trained athletes experienced greater improvements in exercise performance following acute caffeine ingestion than untrained participants, regardless of daily caffeine supplementation. This finding supports some previously published research, ¹⁶ and contradicts other findings.^4,8,33,39 One of the reasons that may explain this difference in findings is that the untrained subjects used in the studies by Astorino et al, ⁴ O’Rourke et al, ³⁹ Boyett et al, ⁸ and Jodra et al ³³ were physically active, while our untrained subjects were inactive. Our results demonstrate that the ergogenicity of caffeine in trained people increased only after 4 weeks of exercise training at the mid-test and then remained constant at the post-test. This observation suggests that a certain level of physical fitness is sufficient for the induction of increased caffeine ergogenicity and that a higher level of fitness will not lead to higher responses. It should be noted that this statement may not apply to elite athletes. In addition, most studies did not report habitual caffeine consumption between different training status groups,^9,16,33,39 which may have influenced the results. It has been suggested that the difference between trained and untrained people may be that trained athletes likely have the mental discipline to exercise long or hard enough to benefit more from the caffeine stimulus, or that their muscles and other tissues are more responsive, which might explain why trained athletes respond better to caffeine. ²⁴

In addition to the ones already mentioned, this study has other limitations that should be considered when interpreting the results. We used a moderate dose of caffeine (3 mg/kg) as a supplement to induce tolerance, and supplementation at higher doses and/or longer periods may produce different responses. Future studies should address whether habitual caffeine intake at higher doses (eg, ≥6 mg/kg) and/or longer periods (eg, several years) could affect the speed of the habituation process as well as the subject’s responses to the short-term caffeine withdrawal and the use of pre-exercise dosage greater than that habitually consumed. Furthermore, the 3-km time trial running and Wingate tests were used to evaluate exercise performance in the current study. If the tests required more motor skills, the interplay effect of fitness status with caffeine might be more pronounced. Moreover, the current evidence suggests that caffeine consumption might cause several side effects, especially in people with low habitual caffeine intake.^5,23 Although our participants were naïve consumers, likely side effects after consuming 3 mg/kg caffeine were ignored, which can be considered a limitation of this study. However, 11 participants in the PLA group, 9 participants in the PLAEXE group, and 1 participant in the CAF group exhibited side effects such as headaches, nervousness, tachycardia and heart palpitations, muscle soreness, nausea, and gastrointestinal problems following 6 mg/kg caffeine ingestion. In 7 of them, exercise performance was partially impaired. Finally, the results of this study were obtained in young men, and factors such as sex, age, and genetic differences may affect these findings, which should be taken into account when interpreting results.

Conclusion

These results suggest that the ergogenic effects of caffeine are enhanced in trained athletes. Habitual caffeine consumption in moderate doses leads to some degree of tolerance. However, a pre-exercise increase in caffeine dosage in trained people and short-term caffeine withdrawal in both trained and untrained people could compensate for the reduced ergogenicity of the substance. It appears that habituation to caffeine and training status could partially influence the ergogenic effects of caffeine on exercise performance.