Effects of Different HIIT Work-to-Rest Ratios on Targeted Metabolomics Profiles and Muscle Damage in Football Players

Background/Objectives: High-intensity interval training (HIIT) improves athletic performance, but the effects of different work-to-rest ratios on amino acid metabolism and recovery remain unclear. This study aimed to compare the acute effects of two HIIT protocols (HIIT1:1 and HIIT1:2) on muscle damage biomarkers, neuromuscular performance, perceived muscle soreness, heart-type fatty acid-binding protein (H-FABP), and comprehensive serum amino acid profiles in trained football players. Methods: Eighteen trained male football players completed two randomized crossover HIIT protocols separated by a washout period. Blood samples and performance assessments were obtained before exercise, immediately after exercise, and at 6, 24, 48, and 72 h of recovery. Serum concentrations of 41 amino acids were quantified using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Blood biomarkers, H-FABP, countermovement jump performance, and perceived muscle soreness were simultaneously evaluated. Time-course responses, area-under-the-curve (AUC) analyses, and correlation analyses were performed. Results: Both HIIT protocols induced transient increases in muscle damage biomarkers and perceived muscle soreness while reducing neuromuscular performance during the early recovery period. Numerous amino acids demonstrated significant temporal alterations, including alanine, arginine, glutamine, glutamic acid, valine, phenylalanine, tyrosine, taurine, methionine, lysine, and several metabolites associated with antioxidant defense, collagen turnover, membrane remodeling, and nitrogen metabolism. Correlation analyses further revealed strong associations between specific amino acids and biomarkers of muscle damage, cardiac stress, and neuromuscular performance. Conclusions: Different HIIT work-to-rest ratios elicited comparable overall physiological stress but distinct metabolic recovery profiles. Amino acid profiling may complement conventional biomarkers in monitoring muscle damage, recovery kinetics, and individualized training responses in football players.