Simple SummaryIn our study, we investigated how Beagle dogs that had not been transported before respond to transport by car. The stress-associated hormone cortisol, heart rate, white blood cells and the dogs’ behavior were analyzed. We hypothesized that transport is perceived as a stressor but dogs get used to it with repeated transports. The dogs were studied in their kennels, in a transport cage placed in a stationary vehicle and during repeated one and two-hour transports. Cortisol concentration increased during transports, and this increase was also evident when dogs were transported repeatedly. Additionally, changes in white blood cells indicated a stress response. Heart rate increased at the beginning of transports, independent from the dogs’ transport experience. During transports but also in the stationary vehicle, dogs were mostly sitting, and time spent standing decreased during experiments. Licking the mouth was the most frequent behavior during transports, but not in the stationary car. In conclusion, a transport-induced stress response was evident in dogs. There was no habituation with repeated transports, and transported dogs may suffer from motion sickness. This study aimed to characterize the response of transport-naïve dogs to one and two-hour road transports based on cortisol in saliva and blood plasma, heart rate, heart rate variability (HRV), neutrophil to lymphocyte (N/L) ratio and behavior. Two persons familiar to the dogs were present during transports and control experiments. We hypothesized that transport elicits a stress response, which decreases with repeated transports. Beagle dogs were allocated to three groups (n = 6 each). Group 1 served as control in the stable in week 1 and was transported for one hour in weeks 2, 3 and 4. Groups 2 and 3 served as controls in a non-moving vehicle and in the stable, respectively, in week 2. All three groups were transported for two hours in week 6. Cortisol concentration increased during transports (p < 0.001), and this increase remained constant with repeated transports. Cortisol release during two-hour transports was not affected by transport experience. Cortisol concentration increased twofold in plasma and eightfold in saliva, indicating an increase in free cortisol. The N/L ratio increased during transport (p < 0.05). Heart rate increased at the beginning of transport while HRV decreased (p < 0.001). Heart rate and HRV neither differed among weeks nor between animals with different transport experience. During transports, but also in the stationary vehicle, dogs were mostly sitting, and time spent standing decreased during experiments (p < 0.001). Licking the mouth was the most frequent behavior during transports but not in the stationary vehicle (p < 0.01). In conclusion, a transport-induced stress response was evident in dogs. There was no habituation with repeated transports, and transported dogs may suffer from motion sickness.