Purpose Caffeine is a stimulant with well-recognized performance and metabolic benefits, however, there is a lack of studies investigating the time-of-day influence in the properties of caffeine to enhance fat oxidation in women. Thus, the aim of the present study was to evaluate the influence of the time of the day on the effect of caffeine on the maximal rate of fat oxidation during aerobic exercise in trained women. Methods Fourteen female athletes (25.5 ± 7.1 years) took part in a randomized, crossover, double-blind study. All participants undertook four different experimental trials combining the ingestion of 3 mg/kg caffeine and a placebo either in the morning (8.00–10.00 h) and in the evening (17.00–19.00 h) realizing an incremental test on a cycle ergometer with 3 min stages at workloads from 30 to 70% of maximal oxygen uptake (VO 2max ). Substrate oxidation rates were measured by indirect calorimetry. In each trial, the maximum rate of fat oxidation (MFO) and the intensity that elicited MFO (Fatmax) were measured. Results In comparison to placebo, MFO was significantly higher with caffeine both in the morning (0.24 ± 0.13 vs 0.30 ± 0.14 g/min; p  < 0.001; ES = 0.79) and in the evening (0.21 ± 0.08 vs 0.28 ± 0.10 g/min; p  = 0.002; ES = 0.72). No time-of-day effect on the capacity of caffeine to increase MFO was found (all p  = 0.336) Conclusion The intake of 3 mg/kg of caffeine increased the use of fat as a fuel during exercise independently of the time-of-day in trained women. Trial registration The study was registered in ClinicalTrials.gov with the following ID: NCT05880186 by 15 May 2023.