Muscle blood flow (MBF) and muscle oxygen saturation (SmO2) were measured at eight locations (four proximal, four distal) over a 4×8 cm2 area of the vastus lateralis at rest and immediately after isometric, maximal quadriceps contraction using multichannel, frequency-domain, near-infrared spectroscopy. A venous occlusion was applied 20 s before the end of the exercise, so that the venous-occlusion-induced increase in total hemoglobin was recorded without any delay after the end of the exercise. Therefore, we were able to investigate the relationship between the exercise-induced changes in vastus lateralis MBF and SmO2. After exercise, MBF increased significantly at each measured location. Comparing the MBF values measured at the end of exercise in the proximal and distal regions, we observed that only one proximal region had a significantly higher MBF than the corresponding distal one. The maximum desaturation measured during exercise was positively correlated with the postexercise to pre-exercise MBF ratio in both the proximal (P = 0.016) and distal (P = 0.0065) regions. These data confirm that frequency-domain tissue oximeters are noninvasive, powerful tools to investigate the spatial and temporal features of muscle blood flow and oxygenation, with potential applications in areas of pathophysiology.