The aim of the study was to analyze motor unit conduction velocity at varying force of the vastus medialis muscle during sustained contraction. Surface (8-electrode array) and intramuscular (two wire electrodes) EMG signals were recorded from the distal part of the dominant vastus medialis muscle of ten healthy male subjects. The subjects sat on a chair with the knee 90° flexed and performed seven 180-s long contractions at forces in the range 2.5–30% of the maximal voluntary contraction force. For each force level, the discharge patterns of the newly recruited motor units with respect to the previous force level were identified from the intramuscular recordings and used as trigger for averaging the surface EMG signals. Motor unit conduction velocity was estimated from the averaged surface EMG. Average discharge rate at which motor units were analyzed was the same for each force level (mean ± SD, 8.3 ± 0.8 pulses per second). Motor unit conduction velocity at the beginning of the contraction and its rate of change over time increased with force (P < 0.05). Conduction velocity at the beginning of the contraction estimated from the interference surface EMG (4.44 ± 0.66 m/s) and from single motor units (4.75 ± 0.56 m/s) were positively correlated (R ² = 0.46; P < 0.0001) but significantly different (P < 0.05). The results indicate that single motor unit conduction velocity and its rate of change during sustained contraction, assessed at a fixed discharge rate, depend on force level.