The study aimed to identify and quantify the metabolites profile and metabolic pathways in human muscle tissue engaged during exhaustive high‐intensity cycling exercise. Seven healthy physically active men performed a graded exercise test and an exhaustive supramaximal effort at 115% of maximal aerobic power with muscles biopsies performed in rest and immediately after exhaustion for quantifying of muscle metabolites changes by 1H‐NMR spectroscopy. The time until exhaustion (tlim) recorded was 224.7 ± 35.5 s whereas the muscle pH at exhaustion was 6.48 ± 0.05. A total of 54 metabolites were identified and quantified. The most enriched and impacted pathways included: beta oxidation of very long chain fatty acids, mitochondrial electron transport chain, alanine aspartate, and glutamate metabolism, citric acid cycle, arginine biosynthesis, propanoate metabolism, threonine and 2‐oxobutanoate degradation and pyruvate metabolism. In addition, the muscle concentrations in Post exercise, compared to Pre increased significantly (p < 0.0398) for fumarate (42.0%), succinate (101.2%), glucose (249.7%), lactate (122.8%), O‐acetylcarnitine (164.7%), glycerol (79.3%), AMP (288.2%), 2‐oxobutyrate (121.0%), and methanol (58.5%), whereas decreased significantly (p < 0.010) for creatine phosphate (−70.2%), ADP (−56.5%), carnitine (−33.5%), and glutamate (−42.3%). Only the succinate was significantly correlated with tlim (r = −0.76; p = 0.0497). Besides the classical expected contribution of glycolytic and phosphagen energetic pathways, it was demonstrated that the high‐intensity exercise is also associated with pathways indicatives of amino acid and fatty acid oxidation metabolisms, highlighting the inverse relation between changes in the intramuscular succinate levels and tlim.