Purpose: To examine the effects of training on the HPA axis using two new noninvasive tools: salivary cortisol response to awakening and overnight urinary cortisol and cortisone excretion, and on the sympathoadrenal system using overnight catecholamines excretion. To dissociate the effects of training to those of seasonal hormonal variations, endurance-trained men were compared with sedentary men. Methods: Nine untrained (UT) men and 10 triathletes were followed during a 10-month season. Clinical (total score of fatigue, total training load, and performances during the competition period) and hormonal parameters (overnight excretion of glucocorticoids and catecholamines, increment of saliva cortisol response to awakening) were measured. Results: Significant seasonal variations in overnight urinary glucocorticoids (decreased in June) and catecholamines (increased in June) concentrations and in saliva cortisol response to awakening were depicted in the two groups. Whereas urinary cortisol excretion was similar between both groups, overnight urinary cortisone excretion was significantly higher in triathletes compared with UT men (ANOVA: training effect: F-2,F-45 = 9.50, P = 0.0003), suggesting that during a resting day there is a higher inactivation of cortisol into cortisone in highly trained men. Two triathletes developed an overtraining syndrome and presented an increased urinary cortisol/cortisone ratio (> 1) due to lower cortisone inactivation compared with the triathlete group. Conclusion: When not taken into account, seasonal variations may induce errors in the interpretation of hormonal variations with training. The increased intracellular inactivation of cortisol during the night in endurance-trained men uncovers subtle changes in HPA function during training. We show in this study the interest of noninvasive biological markers of the activity of the neuroendocrine system to monitor the repercussion of training load during longitudinal follow-up of athletes.