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In this preliminary study, time-dependent changes in plasma CK and AST activity, tyrosine (Tyr), 3-methyl-histidine (3mHis), glucose and lactate concentrations were analysed in nine horses under two different conditions. Furthermore, intramuscular concentrations of Tyr, 3mHis and activities of cathepsin B, acid phosphatase (ACP), glucose-6-phosphate dehydrogenase (G6PDH) and mRNA expression of ubiquitin were determined at the same time. After studying the effects of exercise alone, the effects of exercise and feeding of an experimental protein/amino acid (AA) supplement were analysed. Horses were submitted to a total of four standardised exercise tests (SETs) of high intensity. Potential markers of muscle break down were determined prior to, immediately after, 4 and 18 h after exercise. The experiment was subdivided into two consecutive periods of 3 weeks. In each period, two SETs were performed. In the second period, horses were fed with the protein/AA supplement within 1 h after exercise. Significant changes in plasma, intramuscular Tyr levels and mRNA expression of ubiquitin were caused both by time in relation to exercise and by treatment with the protein/AA supplement. The experimental supplement significantly decreased the 4-h post-exercise expression of ubiquitin mRNA in muscle. Only a borderline increase of markers of lysosomal involvement was seen and CK and AST activity generally showed their normal post-exercise patterns. A clear post-exercise reduction of this CK activity, however, was not observed after supplementation with the protein/AA mixture. The current findings indicate that horses might benefit from protein and AA supplementation directly after training by decreasing post-exercise proteolysis. The results support that further studies should be performed to characterize changes in equine protein metabolism caused by exercise including underlying molecular mechanisms.