This project was designed to assess the effects of time of day and training status on the benefits of caffeine supplementation for cycling performance. Twenty male subjects (Age, 25 years; Peak oxygen consumption, 57 mL·kg⁻¹·min⁻¹) were divided into tertiles based on training levels, with top and bottom tertiles designated as ‘trained’ (n = 7) and ‘untrained’ (n = 7). Subjects completed two familiarization trials and four experimental trials consisting of a computer-simulated 3-km cycling time trial (TT). The trials were performed in randomized order for each combination of time of day (morning and evening) and treatment (6mg/kg of caffeine or placebo). Magnitude-based inferences were used to evaluate all treatment effects. For all subjects, caffeine enhanced TT performance in the morning (2.3% ± 1.7%, ‘very likely’) and evening (1.4% ± 1.1%, ‘likely’). Both untrained and trained subjects improved performance with caffeine supplementation in the morning (5.5% ± 4.3%, ‘likely’; 1.0% ± 1.7%, ‘likely’, respectively), but only untrained subjects rode faster in the evening (2.9% ± 2.6%, ‘likely’). Altogether, our observations indicate that trained athletes are more likely to derive ergogenic effects from caffeine in the morning than the evening. Further, untrained individuals appear to receive larger gains from caffeine in the evening than their trained counterparts.