Hamstring injuries are common in field-based sports and reinjury rates are high. Recent evidence suggests hamstring injuries often occur during accelerative running, but investigations of hamstring mechanics have primarily considered constant-speed running. Thus, our objective was to compare hamstring lengths and velocities between accelerative running and constant-speed running. We recorded videos of 10 participants during 6 accelerative running trials and 6 constant-speed running trials. We used OpenCap to estimate body segment kinematics and a 3-dimensional musculoskeletal model to compute peak length and step-average lengthening velocity of the biceps femoris (long head) muscle-tendon unit. We compared running conditions using linear mixed models with running speed as the independent variable. At running speeds below 75% of top speed, accelerative running resulted in greater peak lengths than constant-speed running. For example, the peak hamstring muscle-tendon length when a person accelerated from running at only 50% of top speed was equivalent to running at a constant 88% of top speed. Lengthening velocities were greater during accelerative running at all running speeds. Differences in hip flexion kinematics drove the greater peak lengths and lengthening velocities observed in accelerative running. Hamstrings are subjected to longer lengths and faster lengthening velocities in accelerative running than in constant-speed running. This provides a potential biomechanical perspective towards understanding the occurrence of hamstring injuries during acceleration. Our results suggest coaches and sports medicine staff should consider the accelerative nature of running in addition to running speed to quantify exposure to high-risk circumstances with long lengths and fast lengthening velocities of the hamstrings.