Introduction: Hamstring strain injuries typically occur in the proximal biceps femoris long head (BFlh) at high running speeds. Strain magnitude seems to be the primary determinant of strain injury, and may be regulated by muscle activation. In running, BFlh strain is largest in the proximal region, especially at high speeds. However, region-specific activity has not been examined. This study examined the proximal-distal and intermuscular activity of BFlh and semitendinosus (ST) as a function of increasing running speed. Methods: Thirteen participants ran at steady speeds of 4.1 (slow), 5.4 (moderate), and 6.8 m·s (fast) on a treadmill. Region- and muscle-specific EMG activity were recorded at each speed using high-density EMG, and were normalized to maximal voluntary isometric activity. Muscle-tendon unit lengths were calculated from kinematic recordings. Speed effects, regional, and intermuscular differences were tested with Statistical Parametric Mapping. Results: With increasing running speed, EMG activity increased in all regions of both muscles to a similar extent in the clinically relevant late swing phase. Increases in muscle-tendon unit lengths in late swing as a function of running speed were comparatively small. In fast running, EMG activity was highest in late swing in all regions, and reached 115% ± 20% (proximal region, mean ± 95% confidence limit), 106% ± 11% (middle), and 124% ± 16% (distal) relative to maximal voluntary isometric activity in BFlh. Regional and intermuscular EMG patterns were highly individual, but each individual maintained similar proximal-distal and intermuscular EMG activity patterns across running speeds. Conclusions: Running is associated with highly individual hamstring activity patterns, but these patterns are similar across speeds. It may thus be crucial to implement running at submaximal speeds early after hamstring injury for restoration of normal neuromuscular function.