Hamstring strain injuries (HSIs) are prevalent in sports involving high-speed running and most of the HSIs are biceps femoris long head (BFlh) injuries. The primary cause for HSIs during high-speed running remains controversial due to the lack of in vivo measurement of the BFlh muscle behavior during running. Therefore, the purpose of this study was to quantify the muscle-tendon unit (MTU) and fascicle behavior of BFlh during running. Seven college male sprinters (22.14 ± 1.8 years; 177.7 ± 2.5 cm; 70.57 ± 5.1 kg; personal bests in 100m: 11.1 ± 0.2 s) were tested on a motorized treadmill instrumented with two force plate for running at 4, 5, 6m/s. The ground reaction force (GRF), 3D lower limb kinematics, EMG, and ultrasound images of biceps femoris long head (BFlh) in the middle region were recorded simultaneously. BFlh fascicles undergo little length change (about 1 cm) in the late swing phase during running at three submaximal speeds. BFlh fascicle lengthening accounted for about 30% of MTU length change during the late swing phase. BFlh was most active during the late swing and early stance phases, ranging from 83%MVC at a running speed of 4 m/s to 116%MVC at 6 m/s. Muscle fascicles in the middle region of BFlh undergo relatively little lengthening relative to the MTU in the late swing phase during running in comparison to results from simulation studies. These results suggest that there is a decoupling between the fascicle in the middle region and MTU length changes during the late swing phase of running.

• This is the first study to measure the biceps femoris long head muscle fascicle behavior during running in vivo.

• The study found that peak muscle activation of BFlh coincided with peak MTU length, indicating high tensile forces during the late swing phase.

• There seems to be a decoupling between fascicle and muscle-tendon unit length changes during the late swing phase.

• The study's findings are likely applicable to higher running speeds, as previous research shows similar muscle-tendon unit length changes across different speeds.

• The degree of fascial lengthening in the present study differed from the computer simulation and may be related to connective tissue compliance and overestimation of tendon stiffness in the simulation.