Purpose Compare acute muscular responses to unilateral, bilateral, and alternating blood flow restriction (BFR) exercise. Methods Maximal strength was tested on visit one. On visits 2–4, 2–10 days apart, 19 participants completed 4 sets of knee extensions (30% one-repetition maximum) with BFR (40% arterial occlusion pressure) to momentary failure (inability to lift load) using each muscle action (counterbalanced order). Ultrasound muscle thickness was measured at 60% and 70% of the anterior thigh before (Pre), immediately (Post-0), and 5 min (Post-5) after exercise. Surface electromyography and tissue deoxygenation were measured throughout. Results, presented as means, were analyzed with a three-way (sex by time by condition) Bayesian RMANOVA. Results There was a time by sex interaction (BFinclusion: 5.489) for left leg 60% muscle thickness (cm). However, changes from Pre to Post-0 (males: 0.39 vs females: 0.26; BF10: 0.839), Post-0 to Post-5 (males: − 0.05 vs females: − 0.06; BF10: 0.456), and Pre to Post-5 (males: 0.34 vs females: 0.20; BF10: 0.935) did not differ across sex. For electromyography (%MVC), there was a sex by condition interaction (BFinclusion: 550.472) with alternating having higher muscle excitation for females (16) than males (9; BF10: 5.097). Tissue deoxygenation (e.g. channel 1, µM) increased more for males (sets 1: 11.17; 2: 2.91; 3: 3.69; 4: 3.38) than females (sets 1: 4.49; 2: 0.24; 3: − 0.10; 4: − 0.06) from beginning to end of sets (all BFinclusion ≥ 4.295e + 7). For repetitions, there was an interaction (BFinclusion: 17.533), with alternating completing more than bilateral and unilateral for set one (100; 56; 50, respectively) and two (34; 16; 18, respectively). Conclusion Alternating, bilateral, and unilateral BFR exercise elicit similar acute muscular responses.