The lack of research on the reaction of horses to working on mechanical treadmills led to the objective of the thesis being the assessment of behaviour and the analysis of the parameters of pony heart rate variability during subsequent stages and working days on the treadmill as the basis for the description of typical behavioural-emotional response categories. The study subject were nine Felin ponies, which for three consecutive days were being accustomed to working on a mechanical treadmill. The parameters of heart rate variability were measured and behaviour was assessed while stabled, when walking onto the treadmill, during treadmill work and while walking off the treadmill. Based on the results obtained, behavioural-emotional reaction patterns of horses as they became accustomed to working on the treadmill were described. The results were subjected to multivariate analysis of variance (repeated measures), t-Tukey test and Spearman rank correlations. It was found that the proposed method of describing the types of emotional and behavioural responses of ponies indicates the necessity to assess their level of being accustomed to working on the treadmill by means of a parallel observation of their behaviour and measurement of heart parameters. The use of two methods is especially necessary during the stages most difficult for the pony: stepping onto the treadmill and working on the treadmill. It is also worth remembering that ponies should first be accustomed to a new environment, and only later to working on a mechanical treadmill. A three-day period of adaptation to the new environment may be insufficient. Care should be taken especially on the second day of getting accustomed, which can be considered critical. Increased emotional arousal/excitability while getting trained for treadmill work is short-lived, which can be considered to be a positive finding.