Invasive procedures in animals are challenging for veterinary students who may perceive a gynaecological examination of mares as stressful. Simulator-based training may reduce stress. In this study, students received equine gynaecology training 4 times either on horses (group H; n = 14) or a teaching simulator (group SIM; n = 13). One day and 14 days thereafter, their diagnostic skills were tested on horses (skills tests 1 and 2). During the skills tests, the students' stress response was analysed by heart rate, heart rate variability (HRV) parameters SDRR (standard deviation of beat-to-beat [RR] interval) and RMSSD (root-mean-square of successive RR differences), and salivary cortisol. In addition, students answered a questionnaire on their perceived stress. Sympathetic activation with increased heart rate (p < 0.001) occurred in both skills tests. In test 1, this increase was more pronounced in SIM than in H students (time × group p < 0.01). HRV decreased in students of both groups (p < 0.001). In skills test 1, this decrease was more pronounced for SIM than for H students (between groups and time × group p < 0.01 for SDRR and p < 0.05 for RMSSD). High cortisol concentrations before the skills tests may indicate an anticipatory stress response. Subjective stress perception of students was higher in skills test 1 vs 2 (p < 0.01). In skills test 2, H students felt more stressed than SIM students (p < 0.01). Self-assessment thus differed from physiological stress parameters. In conclusion, gynaecological examination of mares evoked a moderate stress response in veterinary students, which was more evident after simulator-based than animal-based training.