Mechanical power is a key performance indicator in long track speed skating. Maximal power output in athletic performance can be achieved when mechanical properties of muscles, such as the force-length relationship, are optimized. The purpose of this study was to determine the in vivo operating range of vastus lateralis (VL) fascicle lengths during speed skating imitation and compare the fascicle lengths to those that define the VL force-length relationship. Sixteen sub-elite long track speed skaters (7 females and 9 males;​ body mass:​ 72.5 [11.5] kg;​ age:​ 22.1 [2.7] years) performed maximal voluntary isometric knee extensions at nine different knee joint positions (20–120°) on the left leg to obtain the maximal vastus lateralis (VL) force-length relationship. Participants then performed a speed skating imitation exercise, the turn-cable, at three progressive perceived efforts (50%, 75%, 100%) to identify the VL fascicle excursion during a complete imitation skating stroke. Fascicle lengths and knee joint angles were examined at initial-contact, peak EMG, and take-off. Fascicles between initial contact and peak EMG covered the descending limb of both the maximal and submaximal force-length relationships while operating over the plateau region from peak EMG to take-off. We conclude that the VL works at sub-optimal length during the gliding phase of skating, but at optimal length for maximal force production during the crucial push-off phase where propulsion is provided.