Habituation indicates a decrement in response to a specific stimulus by repeated exposure to that stimulus. Responding to or ignoring sound stimuli, which are common background distractors, is important for the undisturbed functioning of the organism, efficient animal management and safe handling. We habituated 20 warmblood horses to a set of distracting sounds and assessed the possibility of reversing this process through simple modifications in sound playback. The study involved three stages: habituation (Stage 1) and two trials of dishabituation (Stage 2, 3). In Stage 1, five different sounds of animals, each on a separate day, were repeatedly (every 20 minutes (min)) played during a stay on an experimental paddock. The sounds were selected out of 40 sounds of different origin based on their distracting effect on the horses (e.g., interruption of previous activity, increase in emotional arousal) during the first exposure to them three months earlier. In Stage 2, four new and unknown sounds (futuristic characteristics) were additionally introduced to the set of habituated sounds, whereas in Stage 3, habituated sounds (from Stage 1) were played from an altered location. Behavioral (e.g., disruption of feeding, duration of feeding, locomotion and standing alert) and cardiac (heart rate and heart rate variability) variables were assessed. Generally, horses quickly habituated to the initially distracting sounds, but great variability was observed in the number of needed playbacks (1–7) for each horse. After completion of Stage 1, the horses responded with an increase in vigilance, locomotion, refeeding frequency and a 1-minute increase in mean heart rate to novel but not to known sounds. None of the modifications resulted in a recovery of previously habituated reactions. After introduction of playback modifications, only maximum heart rate (in Stage 2, Stage 3) and time of standing alert (in Stage 3) increased after re-exposure to sounds horses had been habituated to, suggesting some level of vigilance. However, this response was too weak to conclude about dishabituation. The results of the study confirm sound recognition and the fast and persistent audio habituation in horses. © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.