ABSTRACT: Background: Racehorses undergo profound physiological changes in response to training and competition, yet current tools to assess training adaptation and overload remain limited. Traditional biomarkers often fail to reflect the complex molecular dynamics induced by physical effort. This study aimed to identify new plasma biomarkers of exercise adaptation in racehorses using high-throughput proteomics. Objectives: We hypothesized that systematic training and racing efforts induce distinct proteomic signatures in plasma, enabling the identification of novel candidate biomarkers reflecting training status, oxidative stress, inflammation, and metabolic remodeling. Study Design: This was an in vivo longitudinal study using repeated blood sampling in the same horses across different phases of training and racing. Methods: Forty-nine Arabian and Thoroughbred racehorses underwent standardized high-intensity training. Plasma samples were collected at rest, immediately post-exercise, and after recovery, during three phases: early training (T1), mid-season conditioning (T2), and racing (R). A total of 314 samples were analyzed using TMT-based quantitative proteomics and high-resolution mass spectrometry. Protein expression changes were evaluated with statistical correction for multiple comparisons (FDR < 0.05), and functional pathway enrichment was conducted via STRING and ShinyGO. Results: The proteomic response evolved across phases: T1 revealed a broad activation of inflammatory (S100A8/A9), antioxidant (SOD1, catalase), and metabolic proteins (G6PD, PGK1), while T2 showed refined expression of remodeling and redox regulators (decorin, thymosin β4, glutathione S-transferase). The racing phase induced the strongest response, with >100 upregulated proteins involved in energy metabolism, oxidative stress defense, and cytoskeletal adaptation. Several proteins (e.g., S100A8, thymosin β4, prothymosin α, cofilin-1, lipocalins) consistently changed across all phases, indicating their biomarker potential. Conclusions: This study identified a panel of promising plasma biomarkers reflecting exercise adaptation and overload in racehorses. These novel candidates may enhance monitoring of performance, training status, and early signs of overtraining in equine athletes.