In this study we compared substrate oxidation and muscle oxygen availability during sustained intermittent intense and continuous submaximal exercise with similar overall (i.e. work and recovery) oxygen consumption (VO2). Physically active subjects (n = 7) completed 90 min of an intermittent intense (12 s work:18 s recovery) and a continuous submaximal treadmill running protocol on separate days. In another experiment (n = 5) we compared oxygen availability in the vastus lateralis muscle between these two exercise protocols using near-infrared spectroscopy. Initially, overall VO(2) (i.e. work and recovery) was matched, and from 37.5 min to 67.5 min of exercise was similar, although slightly higher during continuous exercise (8%; P < 0.05). Energy expenditure was constant (22.5-90 min of exercise) and was not different in intermittent intense [0.81 (0.01) kJ x min(-1). kg(-1)] and continuous submaximal [0.85 (0.01) kJ x min(-1) x kg(-1)] exercise. Overall exercise intensity, represented as a proportion of peak aerobic power (VO2(peak)), was 68.1 (2.5)% VO2(peak) and 71.8 (1.8)% VO2(peak) for intermittent and continuous exercise protocols, respectively. Fat oxidation was almost 3 times lower (P < 0.05) and carbohydrate oxidation was approximately 1.2 times higher (P < 0.05) during intermittent compared to continuous exercise, despite the same overall energy expenditure. Capillary plasma lactate was constant from 15 to 90 min of exercise, and pyruvate was constant from 15 to 75 min, although both were higher (P < 0.0001, lactate; P < 0.001, pyruvate) during intermittent [5.05 (0.28) mM, 200 (7) microM, respectively] compared to continuous exercise [2.41 (0.10) mM, 114 (4) microM, respectively]. There was no difference between protocols for either plasma glycerol or non-esterified fatty acids. The decrease in muscle oxygenation during work periods of intermittent exercise resulted in a lower nadir oxygenation [54.62 (0.41)%] compared to continuous exercise [58.82 (0.21)%, P < 0.001]. The decline in oxygenation was correlated with treadmill speed (r = 0.72; P < 0.05). These results show a difference in substrate utilisation and muscle oxygen availability during sustained intermittent intense and continuous submaximal exercise, despite a similar overall VO(2) and identical energy expenditure.