Simple SummaryTreadmills have become a popular and important tool in various aspects of equestrianism. Habituation to treadmill locomotion has been investigated mainly in the biomechanic aspect. The behavioral aspect of habituation was seldom described; therefore, it was not clear if the habituation process was the same in horses with different temperaments and emotional responses to the handler. We assessed the results horses got in the novel object test, the handling test, and both positive and negative emotional response tests, and their connection with behavior-related features of the habituation process. The four principal components in the examined horses were identified: “Flightiness,” “Freeziness,” “Curiosity,” and “Timidity”. We found that the features of Flightiness gradually decreased during habituation. A part of them increased again when the horses started a new challenge. Features of Freeziness and Curiosity showed strong stability throughout the whole habituation. Features of Timidity strongly increased when the treadmill was introduced; thus, the challenge was completely changed. The first entrance and work on a treadmill seemed to cause fright responses. We conclude that the habituation process should be adapted to the horse’s temperament and emotionality. Such findings will improve handler safety and lead to increased horse welfare during habituation.A treadmill is an important tool in the equine analysis of gait, lameness, and hoof balance, as well as for the evaluation of horse rehabilitation or poor performance including dynamic endoscopy. Before all of these uses, horses have to be habituated to a treadmill locomotion. We used principal component analysis to evaluate the relationship between aspects of the horse’s temperament and emotional response, and progress in the behavioral habituation to a treadmill. Fourteen horses were tested, by the same familiar handler, using the novel object test, the handling test, and both positive and negative emotional response tests. Then, four stages of gradual habituation of the first work on a treadmill were conducted. Each time, the horse’s behavior was filmed. Data obtained from ethograms and heart rate measurements were tested. Four principal components were identified in examined horses: “Flightiness”, “Freeziness”, “Curiosity”, and “Timidity”. Flightiness was connected with nervousness, agitation by new objects, and easy excitability, and gradually decreased of features during habituation. Timidity was associated with a lack of courage and stress in new situations, and those features strongly increased when the treadmill was introduced. Freeziness and Curiosity features showed strong stability throughout the whole habituation. The results of this study provide evidence for a connection between temperament, emotional response, and habituation process in a horse.
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