The objective of this study was to assess the physiological responses in mature healthy horses prior to an 8 h transport, as well as immediately and 24 h after transportation in a horse truck, under autumn conditions typical for central Poland. The study was conducted on a group of 60 horses of different breed. Horses ranged from 4 to 10 years of age. Venous blood was taken 3 times: Idirectly before loading the horses to the trailer, II -directly after unloading the horses from the trailer, III -after period of 24 h resting. There was no change in the concentrations of urea, cholesterol, creatinine, and magnesium and in the activity of alkaline phosphatase during the three subsequent samplings. Albumin, total protein, alanine aminotransferase, aspartate aminotransferase, total bilirubin, creatine kinase, and triglycerides were mostly influenced by the transport. The obtained results indicate that the transportation of horses alters concentrations of physiological variables of their metabolism.