This study analysed the effect of the sex of the horses as well as the type of their maintenance and use at the centre on the behavioural and physiological expression of short-term social isolation, which was assessed on the basis of heart and respiratory rates. A total of 27 clinically healthy horses were examined. The horses had social contact of various durations, were used with different regularity and were not equally accustomed to isolation. An isolation test was conducted. The heart and respiratory rates were measured at rest and after isolation, and the differences between the values were calculated. Moreover, points were assigned for the effect of isolation. The behaviour in subsequent stages of isolation was also observed. It was found that the sex of the horses, the period spent daily in the paddock in the herd, the regularity of use and habituation to staying in isolation can all be classified as factors that have no effect on the heart rate, respiratory rate or behaviour during short-term isolation. It was suggested that the resting respiratory rate and the effect of isolation could be classified as features that are potentially useful in assessing horses’ adaptability to short-term social isolation.