Aims/hypothesis Targeted metabolomic and transcriptomic approaches were used to evaluate the relationship between skeletal muscle metabolite signatures, gene expression profiles and clinical outcomes in response to various exercise training interventions. We hypothesised that changes in mitochondrial metabolic intermediates would predict improvements in clinical risk factors, thereby offering novel insights into potential mechanisms. Methods Subjects at risk of metabolic disease were randomised to 6 months of inactivity or one of five aerobic and/or resistance training programmes ( n  = 112). Pre/post-intervention assessments included cardiorespiratory fitness ( V ⋅ O 2 peak ), serum triacylglycerols (TGs) and insulin sensitivity (S I ). In this secondary analysis, muscle biopsy specimens were used for targeted mass spectrometry-based analysis of metabolic intermediates and measurement of mRNA expression of genes involved in metabolism. Results Exercise regimens with the largest energy expenditure produced robust increases in muscle concentrations of even-chain acylcarnitines (median 37–488%), which correlated positively with increased expression of genes involved in muscle uptake and oxidation of fatty acids. Along with free carnitine, the aforementioned acylcarnitine metabolites were related to improvements in V ⋅ O 2 peak , TGs and S I ( R  = 0.20–0.31, p  < 0.05). Muscle concentrations of the tricarboxylic acid cycle intermediates succinate and succinylcarnitine ( R  = 0.39 and 0.24, p  < 0.05) emerged as the strongest correlates of S I. Conclusions/interpretation The metabolic signatures of exercise-trained skeletal muscle reflected reprogramming of mitochondrial function and intermediary metabolism and correlated with changes in cardiometabolic fitness. Succinate metabolism and the succinate dehydrogenase complex emerged as a potential regulatory node that intersects with whole-body insulin sensitivity. This study identifies new avenues for mechanistic research aimed at understanding the health benefits of physical activity. Trial registration ClinicalTrials.gov NCT00200993 and NCT00275145 Funding This work was supported by the National Heart, Lung, and Blood Institute (National Institutes of Health), National Institute on Aging (National Institutes of Health) and National Institute of Arthritis and Musculoskeletal and Skin Diseases (National Institutes of Health).