Background Treadmills are often used in research, clinical practice, and training. Biomechanical investigations comparing treadmill and overground running report inconsistent findings. Objective This study aimed at comparing biomechanical outcomes between motorized treadmill and overground running. Methods Four databases were searched until June 2019. Crossover design studies comparing lower limb biomechanics during non-inclined, non-cushioned, quasi-constant-velocity motorized treadmill running with overground running in healthy humans (18-65 years) and written in English were included. Meta-analyses and meta-regressions were performed where possible. Results 33 studies (n = 494 participants) were included. Most outcomes did not differ between running conditions. However, during treadmill running, sagittal foot-ground angle at footstrike (mean difference (MD) − 9.8° [95% confidence interval: − 13.1 to − 6.6]; low GRADE evidence), knee flexion range of motion from footstrike to peak during stance (MD 6.3° [4.5 to 8.2]; low), vertical displacement center of mass/pelvis (MD − 1.5 cm [− 2.7 to − 0.8]; low), and peak propulsive force (MD − 0.04 body weights [− 0.06 to − 0.02]; very low) were lower, while contact time (MD 5.0 ms [0.5 to 9.5]; low), knee flexion at footstrike (MD − 2.3° [− 3.6 to − 1.1]; low), and ankle sagittal plane internal joint moment (MD − 0.4 Nm/kg [− 0.7 to − 0.2]; low) were longer/higher, when pooled across overground surfaces. Conflicting findings were reported for amplitude of muscle activity. Conclusions Spatiotemporal, kinematic, kinetic, muscle activity, and muscle-tendon outcome measures are largely comparable between motorized treadmill and overground running. Considerations should, however, particularly be given to sagittal plane kinematic differences at footstrike when extrapolating treadmill running biomechanics to overground running. Protocol registration CRD42018083906 (PROSPERO International Prospective Register of Systematic Reviews).