It is generally accepted that besides cortisol concentrations, parameters of heart rate variability (HRV) are appropriate indicators of stress in horses. The aim of this study was to determine anticipatory stress in eight Standardbred stallions participating in harness race. Cortisol and HRV responses to a mild exercise performed in training circumstances were compared to a maximal effort exercise performed in real trotting race conditions. Parameters of HRV reflecting vagal (root mean square of the successive differences, RMSSD) and sympathetic nervous system activity (ratio of the low and high frequency component, LF/HF) were recorded before warming up (baseline) and during exercise. Plasma cortisol concentrations were obtained for the following stages of the exercise: before warming up (baseline), after warming up, after the exercise has finished and after a 30-min recovery. Baseline LF/HF ratio was higher before the race compared to the pre-training values (12.0 ± 6.6 vs. 5.9 ± 4.5, P = 0.009), while RMSSD did not show such difference (34.8 ± 15.9 ms vs. 48.0 ± 30.5 ms, P = 0.96). Cortisol level was higher in the case of race for all samples compared to training (P = 0.012). There were no significant differences between plasma cortisol levels obtained for the subsequent stages of race. Horses in the present study showed anticipatory response before race as shown by differences in pre-training (97.3 ± 16.4 nmol/L) and pre-race cortisol levels (171.8 ± 18.7 nmol/L), respectively (P < 0.001). Pre-race HRV only partly confirmed this phenomenon.