: The natural differences in running capacities among rats remain poorly understood, and the mechanisms driving these differences need further investigation. : Twenty male Sprague-Dawley (SD) rats were selected. High and low running capacity rats were identified using Treadmill Exhaustion Tests. Peripheral blood was collected for serum isolation, followed by a metabolomics analysis using LC-MS/MS. Data were preprocessed, and a principal component analysis (PCA) and a partial least squares-discriminant analysis (PLS-DA) were applied to identify metabolic profile differences. Significant metabolites were screened, and a pathway enrichment analysis was conducted using the KEGG database to determine key metabolic pathways. Forty SD rats (equal male and female) were randomly divided into an inosine triphosphate (ITP) group (24.29 mg/kg.bw daily) and a control group. Running capacity was assessed after one week of continuous treatment. : Three independent measurements showed consistent differences in running capacity. A total of 519 differential metabolites were identified, with 255 up-regulated and 264 down-regulated. The KEGG pathway analysis revealed a significant enrichment of the Purine Metabolism pathway (ITP-ATP) in the high running capacity group ( < 0.05). The ITP-treated group exhibited a significantly higher running capacity than the controls ( < 0.05), confirming the efficacy of dietary ITP supplementation. : The running capacity of rats is influenced by the ITP-ATP pathway, and exogenous ITP administration through dietary intervention significantly improves running ability.