Plasma adrenocorticotropin hormone (ACTH), cortisol, heart rate, self-reported state anxiety and total reaction time responses in physically active (n=8) and sedentary (n=11) middle-aged men were compared during performance of a well-learned, psychologically challenging task. The groups were similar in age (active: 39.9±8.4 yr vs. sedentary: 44.4±7.2 yr) and trait anxiety (29.4±5.2 vs. 34.5±12.6), but they had different maximal oxygen consumption relative to lean body weight (68.6±7.1 vs. 44.7±4.6 ml · kg −1 · min −1). During performance of the psychological protocol, the total group had significant increases in heart rate and state anxiety, whereas both ACTH and cortisol remained near baseline levels. ACTH and cortisol were not different between the groups. The sedentary group exercise trained for 4 mo, which resulted in an 18% improvement in maximal aerobic capacity (v̇O 2 max). Exercise intervention did not significantly alter the neuroendocrine response compared to pre-training values or compared to a corresponding control group. These findings suggest that enhanced cardiovascular fitness resulting from exercise training does not affect anterior pituitary-adrenocortical activity during performance of a practiced vigilance task.