Abstract: The aim of this study was to evaluate the behavior and physiologic responses of mares to removal from an established pasture herd and to isolation in a pasture setting for 6 h (Group I, n=5). Responses of mares in Group I were compared to mares that were transported and returned to the herd (Group T, n=5) and to mares moved to the isolation pasture with a companion (Group C, n=5). Behavior was recorded continuously for 6 h on the day before the isolation procedures (baseline, Day 0) and again on the day of the procedure (test, Day 1). Plasma cortisol, white blood cell count (WBC), neutrophil:lymphocyte ratio (N:L), and hematocrit (HCT) were measured once on Day 0 (a.m.) and twice on Day 1 (a.m. and p.m.). Heart rate (HR) was monitored continuously during Day 0 and Day 1. Intradermal response to phytohemagglutinin (PHA) injection was measured 18 h following injection, which was administered at the end of Day 1. Average time spent standing alert increased (P<0.05) in Groups I and C and average time spent grazing decreased (P<0.05) in Group C from Day 0 to Day 1. Also, there was a significant difference between groups (based on a calculated χ2-square value) in the proportion of mares that autogroomed, defecated, urinated, rolled, and whinnied on Day 1. Activity shift rate (ASR) and temperament scores increased significantly in Groups I and C from Day 0 to Day 1 (P<0.05). Plasma cortisol increased significantly in all groups from Day 0 to Day 1, a.m. (P<0.05) and decreased significantly from Day 1, a.m. to Day 1, p.m. (P<0.05). HCT significantly increased in all three groups from Day 0 to Day 1, a.m. (P<0.05). WBC significantly increased in Group T from Day 0 to Day 1, a.m. (P<0.05). N:L ratio significantly increased in Groups I and C from Day 0 and Day 1, a.m. to Day 1, p.m. (P<0.05). A variety of measures did indicate a response to removal from the pasture group, however, the overall, short-term response was minimal. Since the responses of Groups I and C were similar, the effects of isolation versus a novel environment or separation from the established herd could not be differentiated.