We investigated the effects of prior heavy exercise on the spatial heterogeneity of muscle deoxygenation kinetics and the relationship to the pulmonary O₂ uptake (pVO₂) kinetics during subsequent heavy exercise. Seven healthy men completed two 6-min bouts of heavy work rate cycling exercise, separated by 6 min of unloaded exercise. The changes in the concentration of deoxy-hemoglobin/myoglobin (deoxy-[Hb+Mb]) were assessed simultaneously at 10 different sites on the rectus femoris muscle using multi-channel near-infrared spectroscopy. Prior exercise had no effect on either the time constant or the amplitude of the primary component pVO₂, while it reduced the amplitude of the slow component (SC). deoxy-[Hb+Mb] across all 10 sites for bout 2 displayed a shorter time delay (the mean and SD for the subjects: 13.5 ± 1.3 vs. 9.3 ± 1.4 s; P < 0.01) and slower primary component time constant () (9.3 ± 1.3 vs. 17.8 ± 1.0 s; P < 0.01) compared with bout 1. Prior exercise significantly reduced both the inter-site coefficient of variation (CV) of the of deoxy-[Hb+Mb] (26.6 ± 11.8 vs. 13.7 ± 5.6 %; P < 0.01) and the point-by-point heterogeneity (root mean squared error, RMSE) during the primary component in the second bout. However, neither change in the CV for nor for the RMSE of deoxy-[Hb+Mb] correlated with the reduction in the SC in pVO₂ kinetics during subsequent heavy exercise. In conclusion, prior exercise reduces the spatial heterogeneity of the primary component of muscle deoxygenation kinetics. This effect was not correlated with alterations in the pVO₂ response during subsequent heavy exercise.