Spectral analysis of heart rate variability has become a noninvasive standard method for assessment of autonomic nervous system activity in athletes. The effect of exercise training on autonomic regulation of pupillary light reflex is not known. The purpose of this study was to evaluate pupil autonomic function in athletes. We studied 46 highly trained athletes practicing gymnastics, swimming, long-distance running, soccer, and 51 healthy control subjects, using a portable infrared pupillometry. Five left pupil light response curves were recorded for each subject; the 485 pupillogram records were processed by a computer system. The following pupillometric parameters calculated were significantly higher (P<0.05) in runners than in controls: reflex amplitude (2.1 mm; 95% CI, 1.9-2.3 vs. 1.8 mm; 95% CI, 1.7-1.9), mean percent reflex amplitude of initial diameter (34%; 95% CI, 32-37 vs. 30%, 95% CI, 28-31) and mean time at which pupil redilated 75% of reflex amplitude (2.15 s; 95% CI, 1.99-2.31 vs. 1.86 s; 95% CI, 1.78-1.93). Sex, age, height, weight, body mass index and years of sports practice had no significant influence in the evaluated parameters. The results were consistent with an increased parasympathetic activity and a reduced sympathetic activity of pupillary light reflex in endurance-trained runners, supporting the hypothesis of a generalized “dysautonomy” associated with this type of training.