Important physiological quantities for investigating muscle hypertrophy include blood oxygenation, cell swelling, and changes in blood flow. The purpose of this study was to compare the acute changes of these parameters in human skeletal muscle induced by low-load (20% 1-RM) blood flow-restricted (BFR-20) knee extensor exercise compared with free-flow work-matched (FF-20WM) and free-flow 50% 1-RM (FF-50) knee extensor exercise using multimodal magnetic resonance imaging (MRI). Subjects (n = 11) completed acute exercise sessions for each exercise mode in an MRI scanner, where interleaved measures of muscle R2 (indicator of edema), [Formula: see text] (indicator of deoxyhemoglobin), macrovascular blood flow, and diffusion were performed before, between sets, and after the final set for each exercise protocol. BFR-20 exercise resulted in larger acute decreases in R2 and greater increases in cross-sectional area than FF-20WM and FF-50 (P < 0.01). Blood oxygenation decreased between sets during BFR-20, as indicated by a 13.6% increase in [Formula: see text] values (P < 0.01)), whereas they remained unchanged for FF-20WM and decreased during FF-50 exercise. Quadriceps blood flow between sets was highest for the heavier load (FF-50), averaging 305 mL/min, and lowest for BFR-20 at 123 ± 73 mL/min until post-exercise cuff release, where blood flow rates in BFR-20 exceeded both FF protocols (P < 0.01). Acute changes in diffusion rates were similar for all exercise protocols. This study was able to differentiate the acute exercise response of selected physiological factors associated with skeletal muscle hypertrophy. Marked differences in these parameters were found to exist between BFR and FF exercise conditions, which contribute to explain the anabolic potential of low-load blood flow restricted muscle exercise.NEW & NOTEWORTHY Acute changes in blood flow, diffusion, blood oxygenation, cross-sectional area, and the "T2 shift" are evaluated in human skeletal muscle in response to blood flow-restricted (BFR) and conventional free-flow knee extensor exercise performed in an MRI scanner. The acute physiological response to exercise was dependent on the magnitude of load and the application of BFR. Physiological variables changed markedly and established a steady state rapidly after the first of four exercise sets.