Abstract: Holstein steer calves (n = 32; 156 {+/-} 33.2 kg average BW) were used to evaluate the duration of restraint and isolation stress (RIS) on endocrine and blood metabolite status and the incidence of dark-cutting LM. Calves were blocked by BW and assigned randomly within blocks to one of four stressor treatments: unstressed controls (NS) or a single bout of RIS for 2, 4, or 6 h. Venous blood was collected via indwelling jugular catheters at 40, 20, and 0 min before stressor application and at 20-min intervals during RIS. Unstressed calves remained in their home stanchions and, except for blood sampling, were subjected to minimal handling and stress. Serum cortisol and plasma lactate concentrations were increased (P <0.01) during the first 20 min after RIS application, and remained elevated throughout the 6 h of RIS. Plasma concentrations of glucose and insulin were greater (P <0.05) in RIS calves than in NS calves after 80 and 100 min of stressor application, respectively; however, RIS did not (P >0.80) affect plasma NEFA concentrations. Calves were slaughtered within 20 min of completion of RIS, and muscle samples were excised from right-side LM at 0, 0.75, 1.5, 3, 6, 12, 24, and 48 h after exsanguination for quantifying LM pH, and glycogen and lactate concentrations. The pH of the LM from calves subjected to 6 h of RIS exceeded 6.0, and was greater (P <0.05) at 24 and 48 h postmortem than the pH of NS calves or calves subjected to 2 or 4 h RIS. Muscle glycogen concentrations did not differ (P = 0.16; 25.58, 10.41, 13.80, and 14.41 {micro}mol/g of wet tissue weight for NS and 2-, 4-, and 6-h RIS, respectively), and LM lactate concentrations tended to be lower (P = 0.08) in calves subjected to 6 h of RIS. At 48 h after exsanguination, the LM from calves subjected to 6 h of RIS had more (P <0.05) bound and less (P <0.05) free moisture than did the LM from NS calves or calves subjected to 2 or 4 h of RIS. Additionally, the LM from RIS calves was darker (lower L* values; P <0.05) than the LM of NS calves. Visual color scores for the LM were greatest (P < 0.05) for calves subjected to 6 h of RIS and least (P <0.05) for NS calves. Subjecting lightweight Holstein calves to 6, 4, and 2 h of RIS resulted in six (75%), two (25%), and two (25%) carcasses characteristic of the dark-cutting condition, respectively. There were no dark-cutting carcasses produced from NS calves. Thus, RIS may be a reliable animal model with which to study the formation of the dark-cutting condition. N1 -