Time-resolved near-infrared spectroscopy was used to quantify tissue oxy- and deoxyhemoglobin concentrations ([HbO2] and [HbR]) and O2 saturation (stO2) in the oblique fibers of the vastus medialis muscle and brain prefrontal cortex during knee extension with and without blood flow restriction (BFR).Six young healthy males performed three sets of knee extensions on a dynamometer (50% one-repetition maximum) separated by 90-s rest periods in three conditions: 1) until fatigue without BFR (fatigue), 2) until fatigue with BFR (100 mm Hg cuff constriction around thigh (BFR)), 3) same number of repetitions from condition 2 without BFR (matched). Each condition was performed on a separate visit.BFR was associated with higher [HbR] at the oblique fibers of the vastus medialis muscle (rest 1: 57.8 (BFR) vs 35.0 μM (matched); P < 0.0001) and a significantly lower stO2 during recovery periods between sets (7.5%-11.2 % lower than non-BFR conditions for rest 1 and 2, P < 0.0001). Using a piecewise linear spline method, a spike in [HbR] was observed before the onset of HbR clearance during recovery, causing HbR clearance to begin at a higher concentration (81 (BFR) vs 62 μM (matched), P = 0.029). [HbO2] kinetics during recovery were also affected by BFR, with longer duration (BFR, 51 s; matched, 31 s; P = 0.047) but lower rate of increase (BFR, 58 μM·min; matched, 89 μM·min; P = 0.004) during recovery. In the prefrontal cortex, BFR was associated with increased [HbR], diminished increase in [HbO2], and higher subjective exertion.These findings yield insight into possible physiological mechanisms of BFR and suggest a role of time-resolved near-infrared spectroscopy in monitoring and optimization of BFR exercise on an individual basis.