Mitochondrial pyruvate dehydrogenase (PDH) regulates the delivery of carbohydrate-derived substrate to the mitochondrial tricarboxylic acid cycle and electron transport chain. PDH activity at rest and its activation during exercise is attenuated following high-fat (HFAT) compared with high-carbohydrate (HCHO) diets. Given the reliance on carbohydrate-derived substrate early in transitions to exercise, this study examined the effects of HFAT and HCHO on phase II pulmonary O2 uptake (V̇o2p) kinetics during transitions into the moderate-intensity (MOD) exercise domain. Eight active adult men underwent dietary manipulations consisting of 6 days of HFAT (73% fat, 22% protein, 5% carbohydrate) followed immediately by 6 days of HCHO (10% fat, 10% protein, 80% carbohydrate); each dietary phase was preceded by a glycogen depletion protocol. Participants performed three MOD transitions from a 20 W cycling baseline to work rate equivalent to 80% of estimated lactate threshold on days 5 and 6 of each diet. Steady-state V̇o2p was greater ( P < 0.05), and respiratory exchange ratio and carbohydrate oxidation rates were lower ( P < 0.05) during HFAT. The phase II V̇o2p time constant (τV̇o2p) [HFAT 40 ± 16, HCHO 32 ± 19 s (mean ± SD)] and V̇o2p gain (HFAT 10.3 ± 0.8, HCHO 9.4 ± 0.7 ml·min−1·W−1) were greater ( P < 0.05) in HFAT. The overall adjustment (effective time constant) of muscle deoxygenation (Δ[HHb]) was not different between diets (HFAT 24 ± 4 s, HCHO 23 ± 4 s), which coupled with a slower τV̇o2p, indicates a slowed microvascular blood flow response. These results suggest that the slower V̇o2p kinetics associated with HFAT are consistent with inhibition and slower activation of PDH, a lower rate of pyruvate production, and/or attenuated microvascular blood flow and O2 delivery.