Abstract: Holstein steer calves (n = 25) were used to evaluate the effects of treadmill exercise (TME) on blood metabolite status and formation of dark-cutting beef. Calves were blocked by BW (156 {+/-} 33.2 kg) and assigned randomly within blocks to 1 of 5 TME treatments arranged in a 2 x 2 factorial design (4 or 8 km/h for a duration of 10 or 15 min) with a nonexercised control. Venous blood was collected via indwelling jugular catheters at 10, 2, and 0 min before TME and at 2-min intervals during exercise. Nonexercised steers were placed on the treadmill but stood still for 15 min. Serum cortisol levels, as well as plasma concentrations of glucose, lactate, and NEFA, were similar (P > 0.05) before TME. Serum cortisol concentrations were unaffected (P > 0.05) during the first 6 min of TME, but between 8 and 15 min of TME, cortisol concentrations were greater (P < 0.05) in steers exercised at 8 km/h than those exercised at 4 km/h or controls (speed x time, P < 0.001). Although TME did not affect (P > 0.05) plasma glucose levels, plasma lactate concentrations in steers exercised at 8 km/h increased (P < 0.05) sharply with the onset of the TME treatment and remained elevated compared with steers exercised at 4 km/h or unexercised controls (speed x time, P < 0.001). Exercised steers had the lowest (P < 0.05) plasma NEFA concentrations during the first 6 min of TME compared with unexercised steers; however, NEFA concentrations were similar after 10 and 12 min of TME, and by the end of TME, steers exercised at 8 km/h had greater (P < 0.05) NEFA levels than nonexercised controls or steers exercised at 4 km/h (speed x time, P < 0.001). Even though muscle glycogen levels and pH decreased (P < 0.001) and muscle lactate concentrations increased (P < 0.001) with increasing time postmortem, neither treadmill speed nor TME duration altered postmortem LM metabolism. Consequently, there were no (P > 0.05) differences in the color, water-holding capacity, shear force, or incidences of dark-cutting carcasses associated with preslaughter TME. It is apparent that preslaughter TME, at the speeds and durations employed in this study, failed to alter antemortem or postmortem muscle metabolism and would not be a suitable animal model for studying the formation of the dark-cutting condition in ruminants.