The aim of this work was to evaluate the changes in the concentrations, after two rides different for distance covered, of different amino acids in endurance horses. Blood samples have been collected from horses just before the start, at the top of a steep slope (819 m difference in height) and just at the end of a 32-km endurance ride. A second group, competing in a 72 km endurance ride, has also been sampled immediately before and after the race. In serum samples, the concentrations of alanine, arginine, asparagine, glycine, isoleucine, histidine, leucine, lysine, methionine, ornithine, phenylalanine, tryptophan, tyrosine and valine have been measured by high-performance liquid chromatography (HPLC). anova and t-test have been used to study the differences in the concentrations of the amino acids. The pre-ride concentrations of the free amino acids were different between the two races, except for methionine and leucine. Differences between start and end race have been found for both groups for all the considered parameters except asparagine, isoleucine, leucine and lysine for the 72 km ride. Increases have been recorded for the shorter and decreases for the longer ride in the blood serum concentrations. Significant increases have also been found between the starting sampling and the second, at the top of the slope, only for alanine, arginine, asparagines, phenylalanine and lysine. The ride length has a significant impact on blood serum amino acids mobilization and uptake; in the shorter race the increases stand only for mobilization, whereas in the longer the decrease can be considered the effect of the onset of the amino acids catabolism.