Purpose: The human biarticular hamstrings [semimembranosus (SM), semitendinosus (ST) and biceps femoris long head (BFLH)] have an important role in running. This study determined how hamstrings neuro-mechanical behaviour changed with faster running, and whether differences existed between SM, ST and BFLH. Methods: Whole-body kinematics and hamstrings electromyographic (EMG) activity were measured from seven participants running at four discrete speeds (range: 3.4 ± 0.1 to 9.0 ± 0.7 m/s). Kinematic data were combined with a three-dimensional musculoskeletal model to calculate muscle-tendon unit (MTU) stretch and velocity. Activation duration and magnitude were determined from EMG data. Results: With faster running, MTU stretch and velocity patterns remained similar, but maxima and minima significantly increased. The hamstrings were activated from foot-strike until terminal stance or early swing, and then again from mid-swing until foot-strike. Activation duration was similar with faster running, whereas activation magnitude significantly increased. Hamstrings activation almost always ended before minimum MTU stretch, and it always started before maximum MTU stretch. Comparing the hamstrings, maximum MTU stretch was largest for BFLH and smallest for ST irrespective of running speed, while the opposite was true for peak-to-peak MTU stretch. Furthermore, peak MTU shortening velocity was largest for ST and smallest for BFLH at all running speeds. Finally, for the two fastest running speeds, the amount of MTU stretch that occurred during terminal swing after activation had started was less for BFLH compared to SM and ST. Conclusion: Differences were evident in biarticular hamstrings neuro-mechanical behaviour during running. Such findings have implications for hamstrings function and injury.