Although exercise is a common and potent activator of the hypothalamic-pituitary adrenal (HPA) axis, the effects of exercise on the acute stress response are not well understood. Here, we investigated the effects of short (2wk)- and long-term (8wk) voluntary wheel running on adrenal sensitivity to ACTH stimulation and the acute stress response to restraint in male rats. Diurnal glucocorticoid (GC) patterns were measured on days 7 (all groups) and 35 (8 wk groups). Rats were subjected to 20 minutes of restraint stress on either week 1 or on week 7 of treatment to assess HPA activation. One week later, exogenous ACTH (75 ng/kg) was administered to assess adrenal sensitivity to ACTH. Following this, adrenals were collected and analyzed for key proteins involved in corticosterone (CORT) synthesis. By the end of week one, exercising (E) animals had a 2-fold higher peak diurnal CORT levels compared with sedentary (S) animals (p<0.01). CORT values were not different between groups at week 8. In response to restraint stress at week 2, CORT values in E were ~3-fold greater than in S (p<0.05). No difference was found between E and S rats in the response to, or recovery from, restraint at week 8. During the ACTH challenge at week 2, E demonstrated a ~2.5 fold increase in adrenal sensitivity compared to S, while no difference was found between E and S at week 8. The expression of steriogenic acute regulatory (StAR) protein was found to be ~50% higher in the adrenals in E compared with S at week 2 (P<0.05), but no difference existed between groups at week 8. These results show that volitional wheel running initially causes hyperactivation of the HPA-axis, due to enhanced adrenal sensitivity to ACTH, but that these alterations in HPA activity are completely restored by 8 weeks of training.