We investigated the effects of caffeine on the ammonia and amino acid metabolism of elite soccer players. In this double-blind randomized study, athletes (n = 19) received 5 mg·kg caffeine or lactose (LEx, control) and performed 45 min of intermittent exercise followed by an intermittent recovery test (Yo-Yo IR2) until exhaustion. The caffeine-supplemented athletes were divided into two groups (CEx and SCEx) depending on their serum caffeine levels (<900% and >10,000%, respectively). Data were analyzed by ANOVA and Tukey post hoc test (P < 0.05 was considered to be statistically significant). Caffeine supplementation did not significantly affect the performance (LEx = 12.3 ± 0.3 km·h, 1449 ± 378 m; CEx = 12.2 ± 0.5 km·h, 1540 ± 630 m; SCEx = 12.3 ± 0.5 km·h, 1367 ± 330 m). Exercise changed the blood concentrations of several amino acids and increased the serum concentrations of ammonia, glucose, lactate, and insulin. The LEx group showed an exercise-induced increase in valine (∼29%), which was inhibited by caffeine. Higher serum caffeine levels abolished the exercise-induced increase (∼24%-27%) in glutamine but did not affect the exercise-induced increase in alanine (∼110%-160%) and glutamate (42%-61%). In response to exercise, the SCEx subjects did not exhibit an increase in uremia and showed a significantly lower increase in their serum arginine (15%), citrulline (16%), and ornithine (ND) concentrations. Our data suggest that caffeine might decrease systemic urea by decreasing the glutamine serum concentration, which decreases the transportation of ammonia to the liver and thus urea synthesis.