Non-invasive measures are preferred when assessing animal welfare. Differences in behavioral and physiological responses toward a stressor could be the result of the selection of horses for specific uses. Behavioral and physiological responses of working and Chilean rodeo horses subjected to a handling test were assessed. Five behaviors, number of attempts, and the time to cross a bridge were video recorded and analyzed with the Observer XT software. Heart rate (HR) and heart rate variability (HRV), to assess the physiological response to the novel stimulus, were registered with a Polar Equine V800 heart rate monitor system during rest and the bridge test. Heart rate variability data were obtained with the Kubios software. Differences between working and Chilean rodeo horses were assessed, and within-group differences between rest and the test were also analyzed. Chilean rodeo horses presented more proactive behaviors and required significantly more attempts to cross the bridge than working horses. Physiologically, Chilean rodeo horses presented lower variability of the heart rate than working horses.