Purpose The ergogenic effect of caffeine on exercise of maximum intensity has been well established. However, there is controversy regarding the effect of caffeine on shifting substrate oxidation at submaximal exercise. The aim of this study was to investigate the effect of acute caffeine ingestion on whole-body substrate oxidation during 1 h of cycling at the intensity that elicits maximal fat oxidation (Fatmax). Methods In a double-blind, randomized, and counterbalanced experiment, 12 healthy participants (VO2max = 50.7 +/- 12.1 mL/kg/min) performed two acute experimental trials after ingesting either caffeine (3 mg/kg) or a placebo (cellulose). The trials consisted of 1 h of continuous cycling at Fatmax. Energy expenditure, fat oxidation rate, and carbohydrate oxidation rate were continuously measured by indirect calorimetry. Results In comparison to the placebo, caffeine increased the amount of fat oxidized during the trial (19.4 +/- 7.7 vs 24.7 +/- 9.6 g, respectively;P = 0.04) and decreased the amount of carbohydrate oxidized (94.6 +/- 30.9 vs 73.8 +/- 32.4 g;P = 0.01) and the mean self-perception of fatigue (Borg scale = 11 +/- 2 vs 10 +/- 2 arbitrary units;P = 0.05). In contrast, caffeine did not modify total energy expenditure (placebo = 543 +/- 175; caffeine = 559 +/- 170 kcal;P = 0.60) or mean heart rate (125 +/- 13 and 127 +/- 9 beats/min;P = 0.30) during exercise. Before exercise, caffeine increased systolic and diastolic blood pressure whilst it increased the feelings of nervousness and vigour after exercise (P < 0.05). Conclusion These results suggest that a moderate dose of caffeine (3 mg/kg) increases the amount of fat oxidized during 1 h of cycling at Fatmax. Thus, caffeine might be used as an effective strategy to enhance body fat utilization during submaximal exercise. The occurrence of several side effects should be taken into account when using caffeine to reduce body fat in populations with hypertension or high sensitivity to caffeine.