Abstract: The traditional way to train horses is by the application of negative reinforcement (NR). In the past few years, however, the use of positive reinforcement (PR) has become more common. To evaluate the effectiveness and the possible stressor effect of the 2 training methods, 12 horses showing severe trailer-loading problems were selected and exposed to trailer-loading. They were randomly assigned to one of the 2 methods. NR consisted of various degrees of pressure (lead rope pulling, whip tapping). Pressure was removed as soon as the horse complied. PR horses were exposed to clicker training and taught to follow a target into the trailer. Heart rate (HR) was recorded every 5 seconds and behavior denoting discomfort was observed using one-zero sampling with 10 seconds sampling intervals. Training was completed when the horse could enter the trailer upon a signal, or was terminated after a maximum of 15 sessions. Of the 12 horses, 10 reached the criterion within the 15 sessions. One horse was eliminated from the study because of illness and 1 PR horse failed to enter the trailer. A Mann-Whitney U-test indicated that the horses trained with NR displayed significantly more discomfort behavior per training session than horses trained with PR (NR: 13.26 ± 3.25; PR: 3.17 ± 8.93, P < 0.0001) and that horses in the PR group spent less time (second) per session to complete the training criterion (NR: 672.9 ± 247.12; PR: 539.81 ± 166.37, P < 0.01). A Mann-Whitney U-test showed that no difference existed in mean HR (bpm) between the 2 groups (NR: 53.06 ± 11.73 bpm; PR: 55.54 ± 6.7 bpm, P > 0.05), but a Wilcoxon test showed a difference in the PR group between the baseline of HR and mean HR obtained during training sessions (baseline PR: 43 ± 8.83 bpm; PR: 55.54 ± 6.7 bpm, P < 0.05). In conclusion, the PR group provided the fastest training solution and expressed less stress response. Thus, the PR procedure could provide a preferable training solution when training horses in potentially stressing situations.