The hormonal response to 4 days' walking exercise (totaling 164 km) was examined among 15 healthy males. We hypothesised that submaximal daily, repeated, prolonged exercise may disturb the balance of the adrenal cortex and pituitary-testicular axis, which could be analysed from the concentrations of serum cortisol and testosterone, and with time-resolved immunofluorometric assay (IFMA) of luteinising hormone (LH) and follicle stimulating hormone (FSH). In the adrenal cortex the increased response to acute exercise could be seen after the 1st day. The concentration of testosterone was reduced after the first two exercise sessions and a plateau was reached after the 3rd day of walking. LH decreased during the 2nd and 3rd day by 31% (P = 0.04 and 0.001, respectively) and remained steady on the 4th day. In FSH the suppression was seen all the time after the second exercise session and before the final walk it was reduced by 19% (P = 0.02) compared with the baseline. The acute response of the adrenal cortex and pituitary-gonadal axis (excluding secretion of FSH) disappeared within 4 days of repeated prolonged walking and no dramatic lasting changes occurred despite this major 4-day effort. However, when using the sensitive IFMA, which can detect low concentrations of gonadotropins, secretion of FSH was seen to remain reduced and no stability was seen.