Abstract: In various species, sex, hormonal treatments and oestrous-cycle stage have been shown to affect the animal's response in behavioural tests. Few such studies have been performed in the horse. The main aim of the present study was to investigate whether oestrous-cycle stage affects mares' response to a novel object test and isolation test and, in part, to study whether mares, assumed to suffer from oestrous-related behavioural problems, respond differently in these tests when compared with controls. Twelve mares were tested twice, in oestrus and dioestrus, in a crossover design. Seven behavioural and two heart rate variables were measured for the novel object test and two heart rate variables for the isolation test. Oestrous-cycle stage and whether a mare was classified as a 'problem' mare did not affect the mare's response. However, test order, i.e. the cycle stage a mare was tested in first, affected its reaction. This effect could partly be explained by significant differences between test occasions 1 and 2 in three behavioural variables and one heart rate variable (p < 0.05) in the novel object test. The mares explored the novel object more and had a higher mean heart rate in the first test. Exploring the novel object more could largely be attributed to those mares tested in dioestrus first, perhaps indicating that the mares in oestrus were less receptive to the novel object. The reason for the differences between test occasions could be an effect of learning or habituation.