Abstract: Social isolation in horses may be regarded as a stress factor which implies welfare problems. The aim of the experiment was to examine the effect of different levels of transient social isolation and human presence on the heart rate (HR) in horses. Seven horses were used and the experiment was conducted in a tether-stable without boxes. The HR was recorded electronically, continuously for 40 min during the following test situations: all horses in the stable; experimenter approaches the tested horse, other horses being untied and leaving the stable; tested horse staying alone or in the company of one or two stable-mates; the experimenter attempts to calm the isolated horse; outdoor auditory stimuli from other horses. The HR was significantly higher during the whole period of isolation, and depended on how many horses were left as company for the one tested. The highest HR was observed while other horses were leaving the stable and during perception of outdoor auditory stimuli from others. When in the company of two stable mates, the HR was elevated only while other horses were leaving the stable and during auditory stimuli from outdoors. Human presence evoked a significant increase in HR, probably due to conditioning of horses (expecting to be untied and allowed to join the others), irrespectively whether the tested horse was left alone or with one or two stable-mates.