Bilateral symmetry of the ligaments is a common assumption used as an intrasubject control for clinical diagnosis. The present study investigated the bilateral symmetry of the dorsal Lisfranc ligament (dLL) using ultrasound. Data were acquired from 50 asymptomatic subjects in a seated position at a loaded calf raise machine equipped with a force plate. The testing conditions included low, medium, and high stress at 0° and 15° abducted foot positions. Images of the dLL were captured and measured using a 10.0-MHz ultrasound transducer and custom written MATLAB software, respectively. The data were analyzed using paired <em>t</em> tests to compare the bilateral measurements of the dLL length under all test conditions. The bilateral pooled dLL length was 7.01 ± 1.38 mm and showed a moderate correlation with the foot length and width. No bilateral differences were found in the dLL length under any of the stress loads in the abducted position or under the medium and high stress load in the rectus position. However, the low stress load rectus position demonstrated a significant bilateral difference in the dLL length (<em>p</em> = .005). The smallest bilateral difference was observed at the 15° abducted position under medium stress (measurement error mean −0.062 mm). Our data suggest that the contralateral dLL length can be used as an intrasubject control for clinical purposes. However, we recommend that the dLL length measurements should be taken in weightbearing position with the foot in the abducted position under medium stress (bilateral stance), reducing potential strain-induced asymmetry.