Horse trekking (HT) is having a stroll on a horse along a walking trail in a forest, field, and/or sandy beach. Generally in HT, horses exercise in tandem line outside the riding facilities. Because the leading horse will be confronted with stressors in the forefront, we hypothesized that the leading horse shows higher stress responses than the following one. In order to verify the hypothesis, we compared short-term stress responses between each position in six horses. Exercise consisted of 15 min of ground riding and 45 min of HT with walking and trotting. Heart rate variability was analyzed for 5 min at 30, 60, and 90 min after the exercising period. There was no significant difference in heart rate during exercise between leading and following positions. The high frequency / low frequency power band of heart rate variability, an index of sympathetic nervous activity, after exercise, tended to be higher in the leading position than following one (P < 0.1). The result in this study can suggest that the leading horse was in a higher stressed state than the following horse after HT.