According to the force-length-velocity relationships, the muscle force potential during locomotion is determined by the operating fibre length and velocity. We measured fascicle and muscle-tendon unit length and velocity as well as the activity of the human vastus lateralis muscle (VL) during walking and running. Furthermore, we determined the VL force-length relationship experimentally and calculated the force-length and force-velocity potentials (i.e. fraction of maximum force according to the force-length-velocity curves) for both gaits. During the active state of the stance phase, fascicles showed significantly (p < 0.05) smaller length changes (walking: 9.2 ± 4.7% of optimal length (L₀); running: 9.0 ± 8.4%L₀) and lower velocities (0.46 ± 0.36 L₀/s; 0.03 ± 0.83 L₀/s) compared to the muscle-tendon unit (walking: 19.7 ± 5.3%L₀, −0.94 ± 0.32 L₀/s; running: 34.5 ± 5.8%L₀, −2.59 ± 0.41 L₀/s). The VL fascicles operated close to optimum length (L₀ = 9.4 ± 0.11 cm) in both walking (8.6 ± 0.14 cm) and running (10.1 ± 0.19 cm), resulting in high force-length (walking: 0.92 ± 0.08; running: 0.91 ± 0.14) and force-velocity (0.91 ± 0.08; 0.97 ± 0.13) potentials. For the first time we demonstrated that, in contrast to the current general conception, the VL fascicles operate almost isometrically and close to L₀ during the active state of the stance phase of walking and running. The findings further verify an important contribution of the series-elastic element to VL fascicle dynamics.