BackgroundWalking speed, a key element of gait analysis, is essential for evaluating the biomechanics of the musculoskeletal system and is typically assessed on flat surfaces, such as walkways or treadmills. While many authors have compared the differences and similarities between treadmill and overground walking, no studies have yet investigated the differences between treadmill gait analysis at self-selected speed (SS) and overground simulated speed (OS). The hypothesis is that accurate kinematic measurements depend on selecting the correct gait speed; however, a mismatch between the perceived comfortable treadmill speed and actual overground speed may affect the accuracy of treadmill gait analyses. This study aimed to assess treadmill gait in healthy young adults by comparing the SS with the OS. The objectives were to determine whether participants could match SS with OS on a treadmill, examine sex differences in gait kinematics and spatiotemporal parameters (KSP) at different speeds, and identify which speed better reflects natural gait kinematics.MethodsA total of 60 healthy men and 70 healthy women, aged 22–35 years, participated in this cross-sectional study to investigate the gait kinematics and spatiotemporal differences between the SS and OS. Student’s t-test, Bonferroni adjustment, Cohen’s effect size, and quadratic regression were employed to analyse differences across walking speeds and groups.ResultsA discrepancy between OS and SS was observed in 66.4% of the participants. Our findings revealed that the adjusted R² values for KSP at SS were consistently greater than those at OS, suggesting that SS offers a more robust and accurate representation of gait kinematics, whereas OS is less reliable.ConclusionsThese findings underscore the importance of individualized speed selection in gait analysis, as it significantly impacts the accuracy of kinematic and spatiotemporal measurements. This insight is pivotal for clinicians and researchers to develop more effective rehabilitation strategies and comprehensively understand gait dynamics.