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Plasma amino acid (PAA) concentration in horses vary according to the exercise type. This study evaluated the changes in PAA levels and the associated metabolites, urea and ammonia, following short-duration, high-intensity cross-country exercise in eventing horses. Twenty eventing horses participated in 55 rides at 14 international competitions (2* to 4* levels) across five venues in Germany and Poland. Blood samples were collected at four timepoints: before exercise (TP0), at 10 min (TP1), and at 30 min (TP2) post-exercise, as well as in the morning on the day after the competition (TP3). A total of 23 different PAAs and two metabolites (ammonia and urea) were analyzed. PAA concentration difference over time was assessed by a mixed ANOVA. Significant fluctuations were observed in 18/25 parameters. For 21/23 PAAs, levels increased at TP1 and/or TP2, while cysteine concentrations decreased. Concentrations returned to pre-competition levels for 21/23 PAAs by TP3. Proline levels remained elevated (p = 0.002), while those of glycine significantly decreased (p = 0.027) at TP3. Plasma ammonia and urea levels increased at TP1, TP2 and TP3. This study provides foundations for supplementation strategies and can inform future works exploring PAAs' role in performance and training adaptation in eventing horses and their potential as performance-related biomarkers.