It is important to manage potential stress experienced by animals used in teaching veterinary students various clinical procedures so as to not compromise animal welfare standards. In this study the potential stress experienced by mares during transrectal palpation of the reproductive tract by veterinary students was assessed by means of heart rate variability (HRV) and endocrine stress-related indicators (salivary glucocorticoid- and faecal glucocorticoid metabolite concentrations). The technique evaluation and standardisation confirmed that care should be taken when interpreting HRV results as correction factors can have an influence on the HRV indicators and heart rate measures. In addition, the repeatability and reliability of heart rate measures and HRV indicators may differ depending on the environment (unrestricted vs. restricted movement) being assessed. Although endocrine stress-related indicators did not indicate an overall stress response, the autonomic nervous system (ANS) measured by HRV reflected identify short-term variations in autonomic cardiac control during palpation. Furthermore, the most significant shifts towards the sympathetic component were recorded during the first 5 min of palpation and 85 min after the start of palpation. Coactivation of the sympathetic and parasympathetic branches of the ANS in the initial stage of palpation may be attributed to recognition (prediction of outcome) of the procedure. The age and experience of the habituated horses did not influence their stress response. Overall, the 20 min palpation period, restricted to one student, was tolerated well by the mares accustomed to the procedure, but the stress response after 55 min restricted movement was pronounced.