Abstract: The purpose of this study was to investigate the response of horses to confinement and isolation in a stable (indoor individual housing) for the first time using behavioral indices, heart rate, and salivary cortisol concentration. Six naive 2-year-old Australian Stock Horse fillies were examined at 4-hour intervals over 24 hours in an outdoor group paddock followed by 24 hours in indoor individual housing. Behavioral observations and scores and heart rates were recorded and saliva samples were taken at each interval. During stabling, all horses became agitated and demonstrated increased vocalization and movement. Behavioral scores were significantly higher in the indoor individual housing (P < .001). No significant difference in heart rates between the two environments was detected. Mean salivary cortisol did not increase significantly (2 ng/mL ± 1.4 ng/mL in outdoor group paddock vs 2.5 mL ± 1.2 ng/mL in indoor individual housing). No diurnal rhythm in salivary cortisol was evident in either the outdoor group paddock or indoor individual housing. The results of this study highlight that a combination of behavioral and physiological measures allow better understanding of stress, where one measurement may be misleading. First time stabling of horses elicited marked behavioral responses indicative of stress that were not reflected in increased heart rates or salivary cortisol concentrations. The lack of a diurnal cortisol rhythm and the comparatively high basal cortisol concentrations found in the outdoor group paddock environment may imply that the fillies were already stressed; therefore, stabling did not cause further aberrations detectable by salivary cortisol analysis.