Simple SummaryIn athletic horses, the evaluation of the performance levels is of major importance during training to prevent sports injuries. Raman spectroscopy is an innovative technique that allows for a rapid evaluation of biological fluids. The application of Raman spectroscopy on serum samples collected from five Italian Saddle horses subjected to physical exercise confirms the utility of this technique for the assessment of the levels of serum metabolic biomarkers that cause the central and peripheral fatigue that occur in athletic horses during physical exercise. In particular, in two bands ((1300–1360) cm−1and (1385–1520) cm−1), the metabolites involved in the insurgence of fatigue were identified. Lipids and tryptophan were identified in the (1300–1360) cm−1band, and leucine, glycine, isoleucine, lactic acid, tripeptide, adenosine, and beta carotene were identified in the (1385–1520) cm−1band. The area of each evaluated sub-band was used for evaluating changes in the conformational structure of proteins in sera that may depend on exercise. In conclusion, the application of Raman spectroscopy on blood serum samples represents a useful technique for secondary-structure protein identification to investigate the metabolic changes that occur in athletic horses during physical exercise.AbstractThe evaluation of the performance levels in athletic horses is of major importance to prevent sports injuries. Raman spectroscopy is an innovative technique that allows for a rapid evaluation of biomolecules in biological fluids. It also permits qualitative and quantitative sample analyses, which lead to the simultaneous determination of the components of the examined biological fluids. On the basis of this, the Raman spectroscopy technique was applied on serum samples collected from five Italian Saddle horses subjected to a standardized obstacle course preceded by a warm-up to evaluate the applicability of this technique for the assessment of central and peripheral fatigue in athletic horses. Blood samples were collected via jugular venipuncture in a vacutainer tube with a clot activator before exercise, immediately after exercise, and 30 min and 1 h after the end of the obstacle course. Observing the obtained Raman spectra, the major changes due to the experimental conditions appeared in the (1300–1360) cm−1and (1385–1520) cm−1bands. In the (1300–1360) cm−1band, lipids and tryptophan were identified; in the (1385–1520) cm−1band, leucine, glycine, isoleucine, lactic acid, tripeptide, adenosine, and beta carotene were identified. A significant effect of exercise was recorded on all the sub-bands. In particular, a change immediately after exercise versus before exercise was found. Moreover, the mean lactic concentration was positively correlated with the Raman area of the sub-band assigned to lactic acid. In this context, the application of Raman spectroscopy on blood serum samples represents a useful technique for secondary-structure protein identification to investigate the metabolic changes that occur in athletic horses during physical exercise.