In this study, we investigated changes in stress response of young transport-naive horses (n=8) during repeated transport of 3.5 hours duration on the road. To analyse the activity of suprarenal gland, we investigated faecal cortisol metabolites concentrations, immunoreactive salivary concentrations before, during and after transportations. Horses showed at all transports a significant increase in salivary cortisol concentrations (P<0.001), which decreased successive with each transport (P<0.05). Another non-invasive method to detect stress is the measurement of heart rate and heart rate variability. For the recording of the cardiac activity and detection of the heart rate variability a mobile recording system (S810i, POLAR, Kempele, Finland) was used. The midpoints of HRV variables for subsequent intervals of 5- and 30 minutes were determinate and the results compared by statistical analysis. Values differ between methods especially in HRV Parameter SD 2 (P<0.001, P<0.001) from transport 2 to transport 4. Coevally SD 2 values differ in both methods between baseline values. However, the two methods can be suited to investigate differences in HRV values. In response to the first transport, horses showed a significant decrease in mean RR interval during loading and start of transportation (P<0.05). However, the rise in mean RR became less pronounced from transport 1 to transport 4 (P<0.001). Moreover in response to onset of transport 2 to 4 the HRV measure SD 2 (standard derivation 2) increased significant (P<0.001). In general, being transported is an unnatural situation for horses which can lead to a wide range of potential stressors in domestic animals which they would naturally avoid. Nevertheless, with adequate training and experience, many horses easily adapt to being transported in vehicles.