Abstarct: Background and Aim: The use of ergogenic supplements such as caffeine and nitric oxide precursors (e.g., arginine, citrulline, and beetroot juice) is common among athletes seeking to enhance performance and mextabolic efficiency. However, the acute combined effects of caffeine and nitric oxide-enhancing supplements on substrate utilization and nitric oxide response during exhaustive exercise remain insufficiently understood. This study aimed to investigate the acute impact of caffeine and nitric oxide precursors on fuel selection, nitric oxide bioavailability, and biochemical blood responses in trained individuals during exhaustive treadmill exercise. Methods: In a randomized, double-blind, placebo-controlled crossover design, sixteen trained male athletes (VO₂max ≥ 50 ml/kg/min) were assigned to four conditions: placebo (A), caffeine (B; 4 mg/kg body weight), nitrate (C; 300 ml beetroot juice + 3 g arginine + 3 g citrulline), and nitrate + caffeine (D). Following supplement ingestion, participants performed a treadmill-baxsed exhaustive exercise test. Blood samples were collected at baxseline, 60 minutes post-supplementation, and immediately post-exercise to assess serum nitrate, glucose, glycerol, and fat oxidation indices. Results: The acute administration of a combined supplement containing nitric oxide precursors (beetroot juice, L-arginine, and L-citrulline), with or without caffeine, significantly increased serum nitric oxide levels. However, the supplements demonstrated no significant effect on fat oxidation indices (MFO and Fatmax). Furthermore, no significant differences in blood glucose and glycerol levels in response to exercise were observed among the different groups. Conclusion: Although the acute use of this supplement combination effectively elevated nitric oxide levels, this increase did not translate to improved fat or carbohydrate mextabolism during exercise. Therefore, this specific supplement formulation is not recommended for enhancing fat oxidation in the short term.