Stresses imposed on livestock have significant impact on their health and productivity as well as public perceptions of animal welfare. Understanding stress responses in livestock may help refine management procedures and facilitate selection of stress-tolerant animals. In this study, behavioral (chute entry order, chute behavior, and exit velocity), physiological (serum cortisol), and biochemical (kinome) responses were evaluated in cattle ( = 20) subjected to three 5-min restraint periods with weekly intervals. Correlations among stress responses were assessed across all animals as well as for subgroups ( = 4) representing animals consistently displaying a high and low extreme of serum cortisol responses. Across all animals, entry order ( = 0.006) and exit velocity ( = 0.023) were positively correlated with serum cortisol; however, these correlations were not consistently reproducible for the high and low serum cortisol responders. Kinome profiling of peripheral blood mononuclear cells revealed distinct signaling events between the high and low cortisol responders. In particular, kinome profiling revealed significant differences in carbohydrate metabolism and apoptosis that were independently validated. Furthermore, changes in serum glucose levels provided a reliable, inexpensive indicator of serum cortisol levels and often had greater predictive value than cortisol for stress-related behavioral responses. Serum cortisol levels displayed a pattern consistent with sensitization, whereas no habituation or sensitization was observed for serum glucose levels or behavioral responses. Collectively, this investigation provides insight into correlations among physiological, behavioral, and biochemical responses of cattle subjected to a brief restraint that may provide biomarkers for selection of stress-tolerant animals.