Eight mature mares were exercised for 20 min at 60% V̇O2max and to fatigue at 95% V̇O2max. Plasma β-endorphin (EN) concentrations were determined before exercise, after a 10 min warm-up, and at the end of each exercise test Mean ± s.e. β-endorphin concentrations were significantly (P<.05) higher following work at 95% V̇O2max when compared to that at 60% V̇O2max (420.0 ± 102.7 vs. 269.9 ± 30.69 pg/ml). Pre-exercise samples were collected between 1000 and 1500 h. In order to evaluate whether any cyclic changes in EN secretion effects may have influenced results, a subsequent study was completed using 12 horses to assess diurnal fluctuations in EN concentrations, by measuring EN concentration every 2 h for a 24 h period. There was no statistical difference in mean values at any time between 0800 and 2000 h. Whether individual differences in EN concentrations reflected varying degrees of stress associated with the exercise could not be determined. Any possible link between EN concentration and exercise stress in exercising horses awaits clarification.