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Abstract Background The aim of the study was to evaluate the performance of contact heat thermal stimulation in horses at different body sites and under different environmental conditions and different test situations. Five warm-blood horses were equipped with the thermal probe located on the skin of nostril (N), withers (W) or coronary band (C). Skin temperature and reaction temperature (thermal threshold) at each location were measured and percent thermal excursion (% TE = 100 * (threshold temperature - skin temperature)/(cut-out temperature - skin temperature) was calculated. Environmental conditions were changed in partial random order for all locations, so each horse was tested in its familiar box stall and stocks, in the morning and evening and at warm and cold ambient temperatures. Type of reaction to the stimulus and horse’s general behaviour during stimulation were recorded. The stimulation sites were examined for the occurrence of possible skin lesions. Results Skin temperatures were significantly different during warm and cold ambient temperatures at all three locations, but remained constant over repeated stimulation. An obvious response to stimulation before reaching cut-out temperature could be detected most frequently at N and W in boxes during warm ambient temperatures. The most frequent type of reaction to thermal stimulation at the nostril was headshaking (64.6%), skin twitching at the withers (82.9%) and hoof withdrawal at the coronary band (79.2%). Conclusion The outcome of thermal threshold testing depended on ambient temperature, stimulation site and environment. Best results with the WTT2 in horses were obtained at the nostrils or withers in a familiar environment at warm ambient temperatures.