Prolonged training and physical effort in endurance horses can influence the performance of the endurance horses negatively. The negative impact of the event is especially noticeable if venues are demanding, i.e. bad weather conditions or demanding grounds, high altitude and especially in cases of insufficiently fit horses. One of the most important factors that influence equine performance is abundance of oxygen in the blood, which is directly related to the air oxygen content and changes with atmospheric pressure, so that high altitude and low air pressure demand additional work from horses and accelerate fatigue. The authors goal was to investigate the influence of workload in endurance racing on sport horse organism. A total of 18 trained and competing endurance horses enrolled in a 60 km long endurance race organized on Zlatibor Mountain were included in our survey. Due to the difficult track 8 horses were disqualified due to lameness, and high heart rate and respiration. Only 10 horses completed the race and these animals were submitted to basic physical, clinical and laboratory examinations, in order to evaluate fatigue and stress reaction in endurance horses in Serbia. Results of the clinical and laboratory testing obtained show significant changes in all parameters evaluated which we attributed to physical effort in all 10 horses that finished the race in comparison with the values obtained before the race. In conclusion, 10 horses finished the race and were tested, and they indicated significant alterations in physiological parameters showing stress reaction. The evaluated parameters decreased into physiological interval values during the 5 days following the race.