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Effect of treadmill familiarization on knee function in osteoarthritis is not clear. Purpose was to determine whether spatiotemporal characteristics, knee joint biomechanics and muscle activation patterns change as individuals with and without medial compartment knee osteoarthritis familiarize to dual-belt treadmill walking over 6 min.20 individuals with knee osteoarthritis and 20 asymptomatic controls walked at a self-selected speed. Spatiotemporal characteristics, sagittal plane joint motions, sagittal and frontal plane moments and knee joint muscle activation patterns, amplitude normalized to maximum isometric contractions were analyzed. Discrete measures were extracted from each biomechanical waveform and principal component analysis was used to determine knee joint muscle activation patterns. Statistical significance was determined using Analysis of Variance models (alpha = 0.05).Spatiotemporal gait characteristics, knee motion and moment differences were found between groups however no group by time interactions existed and no changes in these variables were found over 6 min of walking. Group differences in muscle activation patterns were found in all muscle activations. Muscle activation amplitude and patterns at minute 5 and 6 were generally lower, less prolonged and more dynamic when compared to minute 1 and 3.Individuals with and without medial compartment knee osteoarthritis familiarized to treadmill walking in a similar manner. Minimal changes to knee biomechanics were found during treadmill familiarization. Five to six minutes of familiarization should be considered for surface electromyography in these populations.Dual belt treadmill familiarization in knee osteoarthritis and asymptomatic controlsGroup effects not affected by treadmill familiarizationNo change in spatiotemporal and gait biomechanics over 6 min of walkingMuscle activation amplitudes and patterns affected by treadmill familiarizationAt least 5 min of dual belt treadmill walking familiarization is recommended.