This study investigated the effects of high-intensity interval training (HIIT) versus work-matched moderate-intensity continuous exercise (MOD) on metabolism and counter-regulatory stress hormones. In a randomized and counter-balanced order, 10 well-trained male cyclists and triathletes completed a HIIT session (81.6±3.7% VO2max; 72.0±3.2% peak power output; 792±95 kJ) and a MOD session (66.7±3.5% VO2max; 48.5±3.1% peak power output; 797±95 kJ). Blood samples were collected before, immediately after, as well as 1 h and 2 h post-exercise. Carbohydrate oxidation was higher (P=0.037; 20%), whereas fat oxidation was lower (P=0.037; -47%) during HIIT vs MOD. Immediately after exercise, plasma glucose (P=0.024; 20%) and lactate (P<0.01; 5.4x) were higher in HIIT vs MOD, whereas total serum free fatty acid concentration was not significantly different (P=0.33). Metabolomics analysis identified and quantified 49 metabolites in plasma, among which, 11 changed after both HIIT and MOD, 13 changed only after HIIT and five changed only after MOD. Notable changes included substantial increases in tricarboxylic acid intermediates and monounsaturated fatty acids after HIIT, and marked decreases in amino acids during recovery from both trials. Plasma adrenocorticotrophic hormone (P=0.019), cortisol (P<0.01) and growth hormone (P<0.01) were all higher immediately after HIIT. Plasma noradrenaline (P=0.11) and interleukin-6 (P=0.20) immediately after exercise were not significantly different between trails. Plasma insulin decreased during recovery from both HIIT and MOD (P<0.01). These data indicate distinct differences in specific metabolites and counter-regulatory hormones following HIIT vs MOD, and highlight the value of targeted metabolomic analysis to provide more detailed insights into the metabolic demands of exercise.