BACKGROUND: Individuals with unilateral transfemoral amputation are prone to developing health conditions such as knee osteoarthritis, caused by additional loading on the intact limb. Such individuals who can run again may be at higher risk due to higher ground reaction forces (GRFs) as well as asymmetric gait patterns. The two aims of this study were to investigate manipulating step frequency as a method to reduce GRFs and its effect on asymmetric gait patterns in individuals with unilateral transfemoral amputation while running. METHODS: This is a cross-sectional study. Nine experienced track and field athletes with unilateral transfemoral amputation were recruited for this study. After calculation of each participant's preferred step frequency, each individual ran on an instrumented treadmill for 20 s at nine different metronome frequencies ranging from - 20% to + 20% of the preferred frequency in increments of 5% with the help of a metronome. From the data collected, spatiotemporal parameters, three components of peak GRFs, and the components of GRF impulses were computed. The asymmetry ratio of all parameters was also calculated. Statistical analyses of all data were conducted with appropriate tools based on normality analysis to investigate the main effects of step frequency. For parameters with significant main effects, linear regression analyses were further conducted for each limb. RESULTS: Significant main effects of step frequency were found in multiple parameters (P < 0.01). Both peak GRF and GRF impulse parameters that demonstrated significant main effects tended towards decreasing magnitude with increasing step frequency. Peak vertical GRF in particular demonstrated the most symmetric values between the limbs from - 5% to 0% metronome frequency. All parameters that demonstrated significant effects in asymmetry ratio became more asymmetric with increasing step frequency. CONCLUSIONS: For runners with a unilateral transfemoral amputation, increasing step frequency is a viable method to decrease the magnitude of GRFs. However, with the increase of step frequency, further asymmetry in gait is observed. The relationships between step frequency, GRFs, and the asymmetry ratio in gait may provide insight into the training of runners with unilateral transfemoral amputation for the prevention of injury.