The mechanisms and underlying causes of bilateral asymmetry among healthy runners of different levels remain unclear. This cross-sectional laboratory study aimed to investigate the effects of running speed and running experience or competitive level on bilateral symmetry during running. Eleven competitive runners, 9 recreational runners and 11 novice runners were recruited in this study. They ran on an instrumented treadmill for 3 min at each of 5 fixed speeds (8, 9, 10, 11 and 12 km/h) in a randomized order. Bilateral asymmetry was evaluated and quantified using symmetry index (SI) of temporal and kinetic parameters. Overall, SI ranged between 0.8% for stride time and 21.4% for vertical average loading rate. Significant speed effects were observed on SI of flight time (p = .012), which was significantly higher at 8 km/h than that of the other 4 speeds (p = .023, 0.005, 0.023 and 0.028, respectively). Group-by-speed interactions were detected on SI in time to peak vertical ground reaction force (p = .032) and vertical average loading rate (p = .002). The competitive runners presented linear reduction in the SI with increasing speed from 8 to 12 km/h (R > 0.94); for the recreational runners, SI changed nonlinearly and presented a roughly U-shaped trend across speeds (R > 0.88); and for the novice runners, changes of SI across speed were inconsistent and dependent on parameters of interest (R > 0.64). Bilateral asymmetry was affected by both running speed and runners' running experience or competitive level. The competitive runners were found to run with a more symmetrical manner with a greater running speed, the recreational runners demonstrated the most symmetrical pattern at the critical speed, whereas the novice runners showed inconsistent trends.