Our laboratory recently showed that six sessions of sprint interval training (SIT) over 2 wk increased muscle oxidative potential and cycle endurance capacity (Burgomaster KA, Hughes SC, Heigenhauser GJF, Bradwell SN, and Gibala MJ. J Appl Physiol 98: 1895–1900, 2005). The present study tested the hypothesis that short-term SIT would reduce skeletal muscle glycogenolysis and lactate accumulation during exercise and increase the capacity for pyruvate oxidation via pyruvate dehydrogenase (PDH). Eight men [peak oxygen uptake (V̇o2 peak) = 3.8 ± 0.2 l/min] performed six sessions of SIT (4–7 × 30-s “all-out” cycling with 4 min of recovery) over 2 wk. Before and after SIT, biopsies (vastus lateralis) were obtained at rest and after each stage of a two-stage cycling test that consisted of 10 min at ∼60% followed by 10 min at ∼90% of V̇o2 peak. Subjects also performed a 250-kJ time trial (TT) before and after SIT to assess changes in cycling performance. SIT increased muscle glycogen content by ∼50% (main effect, P = 0.04) and the maximal activity of citrate synthase (posttraining: 7.8 ± 0.4 vs. pretraining: 7.0 ± 0.4 mol·kg protein −1·h−1; P = 0.04), but the maximal activity of 3-hydroxyacyl-CoA dehydrogenase was unchanged (posttraining: 5.1 ± 0.7 vs. pretraining: 4.9 ± 0.6 mol·kg protein −1·h−1; P = 0.76). The active form of PDH was higher after training (main effect, P = 0.04), and net muscle glycogenolysis (posttraining: 100 ± 16 vs. pretraining: 139 ± 11 mmol/kg dry wt; P = 0.03) and lactate accumulation (posttraining: 55 ± 2 vs. pretraining: 63 ± 1 mmol/kg dry wt; P = 0.03) during exercise were reduced. TT performance improved by 9.6% after training (posttraining: 15.5 ± 0.5 vs. pretraining: 17.2 ± 1.0 min; P = 0.006), and a control group ( n = 8, V̇o2 peak = 3.9 ± 0.2 l/min) showed no change in performance when tested 2 wk apart without SIT (posttraining: 18.8 ± 1.2 vs. pretraining: 18.9 ± 1.2 min; P = 0.74). We conclude that short-term SIT improved cycling TT performance and resulted in a closer matching of glycogenolytic flux and pyruvate oxidation during submaximal exercise.