Quantitative three-dimensional gait analysis has been used to evaluate the loading at the knee (i.e. external knee adduction moment, EKAM) during level ground walking in individuals with knee osteoarthritis (OA). The magnitude of EKAM can be influenced by some factors, such as knee marker position and foot placement angles in static calibration trials, which may lead to inaccurate functional assessments and intervention planning. This study aimed to clarify the effects of knee position during static calibration trials on the evaluation of knee loading during gait in individuals with medial knee OA. Seventeen individuals with medial knee OA completed three different static standing trials; (1) knee flexed at 0 degrees, (2) knee flexed at 15 degrees, and (3) knee flexed at 30 degrees before walking at their self-selected speed. A sixteen-camera three-dimensional VICON gait analysis system with four AMTI force platforms was used to collect the EKAM, knee adduction angular impulse (KAAI), knee joint center (KJC), and other knee kinematic and kinetic variables during gait. A repeated measures ANOVA was used to investigate the differences between conditions. The 1st peak of EKAM, the 1st peak EKAM arm, KAAI, and knee extension moment were significantly increased at the 15-degree and 30-degree conditions in comparison with the 0-degree condition (P < 0.05). Additionally, the knee flexion moment and knee external rotation moment were significantly reduced at the 15-degree and 30-degree conditions in comparison with the 0-degree condition (P < 0.05). All biomechanical variables were influenced by the localization of the KJC during static calibration trials. The changes in knee position during static trials significantly affected the 1st peak EKAM, KAAI, and other knee kinematics and kinetics variables during gait. Therefore, future studies should consider keeping the participants’ knees in a consistent position during static trials between visits, as the variations in knee position could mask or exaggerate the differences between groups and interventions.