Autonomic nervous system tone varies with fitness, training, and conditions such as cardiac disease, hypertension, or overtraining. Normal values of autonomic tone markers and changes over a competition season are incompletely described in eventing horses. The objectives of this study were to describe normal values and changes over a competition season of markers of autonomic tone in competing eventing horses. These values were measured in conjunction with previously reported variables to monitor training and compared with non-competing breed-matched controls. Heart rate variability (HRV), non-invasive blood pressure, splenic volume, pre- and post-exercise hematocrit and cortisol, standardized exercise tests (SETs), and muscle enzyme activities were measured preseason (T1), midseason (T2), and at the peak and/or end of the competition season (T3) in eventing and control horses. Heart rate variability was lower (P < .05) at all times and post-exercise cortisol lower at T2 and T3 in eventing horses compared with controls. Heart rate variability and post-exercise cortisol did not change over the season in any group. Eventers had higher fitness levels during SETs than controls. Non-invasive blood pressure, splenic volume, hematocrit, pre-exercise cortisol, muscle enzyme activities, and weight were not significantly different between groups and did not change over the season. The lower HRV in competing eventers suggests a lower parasympathetic and/or higher sympathetic tone in this group. A lower post-exercise cortisol suggests a decreased stress response in eventers to the SET. Non-invasive blood pressure, splenic volume, and resting or post-exercise hematocrit did not detect differences or changes in autonomic tone in this population.