The objective of this study was to evaluate the impacts of stress challenge duration on metabolic, hematologic, and rumen fermentation parameters of beef steers. Thirty steers (416 ± 19 kg) were used in a randomized complete block design with 2 blocks and 3 treatments. Treatments were intravenous injection of: 1) saline at 0, 24, 48, and 72 h (n = 10; Control); 2) Corticotropin releasing hormone (CRH) and vasopressin (VP) at 0 h and saline at 24, 48, and 72 h (n = 10; Acute); or 3) CRH and VP at 0, 24, 48, and 72 h (n = 10; Chronic). Serum samples were collected at various time points for analysis of serum chemistry and non-esterified fatty acids (NEFA). Whole blood was collected for analysis of complete blood count and ruminal fluid was collected via oral lavage to evaluate volatile fatty acid (VFA) composition. Serum cortisol was greater (treatment × hour; P ≤ 0.01) for cattle receiving acute and chronic than control at 1 h (P < 0.01) and greater for chronic than acute and control at 25, 26, 49, 50, 73, and 74 h (P < 0.01). Similarly, there was a treatment × hour interaction (P ≤ 0.01) for serum glucose concentrations such that cattle receiving acute and chronic had greater glucose at 1 h than control (P < 0.01), and cattle receiving chronic had greater glucose at 25, 49, 50, 73, and 74 h than acute and control (P ≤ 0.04). Serum insulin concentrations were greater (treatment × hour P ≤ 0.01) in chronic and acute than control at 1 h (P < 0.01) and greater for chronic compared to acute and control at 25, 49, and 73 h (P ≤ 0.01). Serum NEFA tended (P = 0.09) to be greater in stressed cattle compared to control. There was a treatment × hour interaction (P = 0.003) for total white blood cell count such that chronic had greater concentration than control at 72 h (P < 0.01). Conversely, monocyte concentration was less (treatment × hour interaction P ≤ 0.01) for chronic than acute and control at 144 h (P < 0.01) and eosinophil concentration was greater (treatment × hour interaction P = 0.02) for chronic than control steers at 48 h (P = 0.02) and greater for chronic than acute at 72 and 144 h (P ≤ 0.03). Minimal differences were observed in VFA concentrations with the exception of acetate (treatment × hour interaction P = 0.05). These results demonstrate that administration of CRH and VP affects complete blood count and serum chemistry, and longer duration of treatment exposure prolongs the physiological responses to a stress challenge.