Abstract: It is widely accepted that transport is stressful for horses, but only a few studies are available involving horses that are transported regularly and are accustomed to transport. We determined salivary cortisol immunoreactivity (IR), fecal cortisol metabolites, beat-to-beat (RR) interval, and heart rate variability (HRV) in transport-experienced horses (N = 7) in response to a 2-d outbound road transport over 1370 km and 2-d return transport 8 d later. Salivary cortisol IR was low until 60 min before transport but had increased (P < 0.05) 30 min before loading. Transport caused a further marked increase (P < 0.001), but the response tended to decrease with each day of transport. Concentrations of fecal cortisol metabolites increased on the second day of both outbound and return transports and reached a maximum the following day (P < 0.001). During the first 90 min on Day 1 of outbound transport, mean RR interval decreased (P < 0.001). Standard deviations of RR interval (SDRR) decreased transiently (P < 0.01). The root mean square of successive RR differences (RMSSD) decreased at the beginning of the outbound and return transports (P < 0.01), reflecting reduced parasympathetic tone. On the first day of both outbound and return transports, a transient rise in geometric HRV variable standard deviation 2 (SD2) occurred (P < 0.01), indicating increased sympathetic activity. In conclusion, transport of experienced horses leads to increased cortisol release and changes in heart rate and HRV, which is indicative of stress. The degree of these changes tended to be most pronounced on the first day of both outbound and return transport.