The aim of the study was to measure the responses of various physiological parameters to the workload by riding of the Lipizzan horses. The study included 6 horses (2 mares, 3 geldings and a stallion), which performed two working tests, the first one in the working and the other one in the accelerated tempo. The working test lasted 30 minutes divided into 10-minute periods in walk, trot and gallop. Prior to each working test, the horses had been installed with a telemetric pulse gauge, which also recorded the cowered distance, which served as a basis for the calculation of the reached in each gate. Before the beginning and after the end of the physical load, we measured breathing frequency, body temperature and skin temperature in different parts of the horse body. We also took blood samples for determination of the hematological parameters, lactate and cortisol concentrations. During the entire working test, we measured the air temperature and humidity. We found that the values of most of the measured parameters and their responses to the physical load in the Lipizzan horses are similar to those of other breeds of horses, reported in the literature. Deviations for some parameters were detected in the investigated horses, which can be linked to the current environmental conditions (nervousness, people, stress) and breed characteristics of the examined horses.