Horses in a herd develop and maintain a dominance hierarchy between all individuals. There are many situations in riding facilities and studs in which horses have to be separated out of a group. The aim of the study was to determine the rate of behaviours, level of locomotor activity and cardiac activity variables in a herd of horses during a short social separation of individuals differently ranked in the dominance hierarchy. Twelve adult Arabian mares were involved. A behavioural test had been performed before the main experiment to determine the rank order of the mares in this social herd. Three tests were performed when a dominant, mixed and submissive three-member group of mares was separated for 10 min. The response of the remaining herd was determined by a rate of behaviours, time of locomotor activity and cardiac parameters. The results of the experiment reveal evident changes towards emotional arousal in the social herd elicited by a short separation of some conspecifics. The herd created by humans preserves the sensitivity to a temporary loss of its members. The response of the remaining herd does not depend strictly on the composition of the separated mares regarding their rank in the dominance hierarchy.