There is growing evidence that reactive oxygen species (ROS) are involved in the muscular damage and soreness that is observed following strenuous or unaccustomed exercise. This study investigated the relationship between delayed onset muscle soreness (DOMS), muscle function and ROS following downhill running using electron spin resonance (ESR) spectroscopy and plasma malonaldehyde (MDA) concentrations. Eight physically active male subjects participated in two trials consisting of 30 min of running at approximately 65% VO(2max) on the flat (FLA) or a 15% downhill (DWN) gradient. Venous blood samples were drawn before, immediately after, and then 24, 48 and 72 h post exercise, and at the same time DOMS and muscle function were assessed. Blood was analysed for markers of ROS, total and differential white blood cell count, and creatine kinase. Muscle function was measured on an isokinetic dynamometer, whilst DOMS was assessed using a visual analogue scale. An increase in ROS, detected via ESR spectroscopy and MDA, was observed following DWN ( P<0.05) but not following FLA. Increased DOMS and loss of muscle function were observed following DWN ( P<0.05) but not following FLA ( P>0.05). DWN resulted in a transient leukocytosis ( P<0.05) occurring immediately post-exercise but returning to pre-exercise levels by 24 h. Although DWN resulted in an increase in ROS production, the increase occurred after the peak decline in muscle function and DOMS, suggesting that there may be a disassociation in the temporal relationship between ROS, loss of muscle function and DOMS.