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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.Markers of autonomic tone were repeatedly measured in eventers.Eventing horses had significantly lower heart rate variability than control horses.Eventing horses had a lower postexercise cortisol.