Objectives. Malalignment, dynamic knee instability, and repetitive physical activity are considered biomechanical risk factors for knee osteoarthritis (KOA), though the correlations among these factors are poorly understood. The purpose of this study was to elucidate the relationship between knee instability and alignment, and to determine the effects of repetitive physical activity on knee instability in patients with KOA.Methods. The study subjects were 68 patients with radiographic tibiofemoral KOA and 68 control subjects. Each participant underwent clinical evaluation, muscle strength test, radiography, and knee instability test. Instability was evaluated before and after repetitive stepping exercise using triaxial accelerometer. Results. Mediolateral acceleration correlated (p<0.01) with two coronal alignments (mechanical axis; HKA and joint line convergent angle; JLCA). Pearson correlation coefficient was small (r=0.23-0.24) before but increased after stepping (r=0.28-0.33). Increased mediolateral acceleration after stepping correlated with JLCA (r=0.37, p<0.001) . There were significant differences in coronal alignments, gait speed, mediolateral acceleration, and accelerations in all directions between the control and KOA groups. Anteroposterior acceleration did not correlate with sagittal knee alignment. Multiple logistic regression analysis identified HKA/JLCA, and increased mediolateral acceleration after stepping as significant diagnostic predictors of KOA.Conclusion. We found a direct relationship between knee instability and knee alignment or repetitive physical activity. Repetitive stepping activity significantly increased mediolateral acceleration in KOA patients, compared to the control. Stepping increased the correlation between mediolateral acceleration and coronal alignment. In knees with large JLCA, repetitive stepping provided much larger mediolateral instability. Our results suggest that, in addition to JLCA, the increase in mediolateral acceleration after repetitive physical activity, possibly contributes to the development of KOA.Trial registrationTan-nan Regional Medical Center TRMC No. 2018-1