Abstract: The aim of the study was to investigate the effect of carrying heavy load for a long time on lipid peroxidation (MDA: malondialdehyde), NO2 (nitrite), NO3 (nitrate), antioxidants (GSH: reduced glutathione, retinol, α-tocopherol) and vitamin D3 in healthy horses. Blood samples from seventeen native 3-5 years age and 450-500 kg live weight Anatolian horses carried a load which comprised at least 30% of their body weight and for 4 h on mountainous terrain (hard working) were evaluated. Blood samples were collected in the morning before the animals started to carrying load and immediately after they finished carrying (working). It is observed that the level of MDA, NO2 and NO3 increased significantly (p<0.05) after working. While GSH concentration, increased after working; levels of retinol, α-tocopherol and vitamin D3 levels decreased significantly (p<0.05). On the other hand, the vitamin D3 levels were affected by hard working as other lipid soluble vitamins. There were a correlation between the physiological response to hard-working and some oxidant markers in healthy-hard working horses. These observations provide evidence that hard-working increases oxygen consumption and cause a disturbance of intracellular pro-oxidant-antioxidant homeostasis.