The behavioural and physiological responses of horses to six hours of isolation was examined. The horses were removed from established pasture herds. The responses of the isolated mares was compared with mares that were transported and then returned to the herd, or moved to an isolated pasture with a companion. The mares were observed the day before the isolation procedures to determine the alterations in behaviour during isolation. The plasma cortisol, white blood cell count, neutrophil:lymphocyte ratio and hematocrit were measured the morning before isolation and in the morning and afternoon on the day of isolation. The heart rate of the mares was monitored continuously during isolation and the day before isolation. Phyohemagglutinin (PHA) was administered the day of isolation. The intradermal response was measured 18 hours following the injection. Mares that were isolated on a pasture with or without a companion significantly increased the amount of time they spent standing alert, while only the mares isolated with a companion decreased the amount of time they spent grazing. The mares that were isolated urinated, rolled and whinnied more than expected and autogroomed less than expected, while the transported mares urinated, rolled and whinnied less than expected and autogroomed more than expected. Mares that were isolated with a companion defecated less than what was expected, while the mares that were transported or isolated alone defecated more than what was expected. The isolated mares and those with a companion were increasingly agitated the day of isolation. The plasma cortisol concentrations and hematocrit rose in all the treatment groups the day of isolation. The peak cortisol concentration was recorded the morning of isolation. The white blood cell count increased in the mares that were transported, while the neutrophil:lymphocyte ratio increased in the isolated mares the day of isolation. The results illustrated that the mares responded both behavioural and physiologically to the isolation from the herd. However, the effects were minimal and short in duration. There does not appear to be a difference in how the mares respond to isolation when they are alone or with a companion, hence it was not possible to determine if it was isolation, the new environment or separation from the herd that caused the behavioural and physiological changes.