Adrenocortical steroid hormones play a major role in the regulation of both action and metabolism of catecholamines. However, there is limited information on the effect of prolonged increase in plasma steroid hormone concentration on the sympathetic nervous system, which is the main source of norepinephrine. Using a double-blind, placebo-controlled cross-over design, we examined 20 male healthy volunteers after acute administration of 40 mg hydrocortisone or matching placebo as well as after a 1-week treatment with this steroid (daily dose, 40 mg) or placebo. Sympathetic nervous system activity was assessed by determination of overnight urinary norepinephrine and epinephrine secretion and by analysis of short-time heart rate variability (HRV) measures in the frequency domain. The low-frequency (LF) component of the HRV spectrum was considered to reflect both sympathetic and parasympathetic modulation and the high-frequency (HF) band vagal activity. HRV testing included the response to a mild cardiovascular challenge, orthostasis. After 1 subject had been excluded because of incompliance with the study medication, the data of 19 subjects were used for further analyses. Prolonged hydrocortisone treatment was associated with a significant shift of relative power from the LF to the HF band in the upright position, resulting in a significant decrease in the LF/HF ratio (placebo 6.77 ± 3.35 and hydrocortisone 4.72 ± 3.27; p < 0.05). No differences between groups were observed after acute steroid administration or after prolonged treatment in the supine position. Neither acute nor chronic hydrocortisone treatment resulted in significant changes in the overnight urinary catecholamine secretion. Our data suggest that prolonged but not acute hydrocortisone administration shifts the autonomic response to an orthostatic challenge towards parasympathetic activity.