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Background: In vivo length change behavior of native medial patellofemoral ligament (MPFL) fibers throughout the range of knee motion has not been reported in vivo., Purpose: To measure the length changes of various fibers of the MPFL and to determine their length change patterns during in vivo passive knee flexion., Study Design: Descriptive laboratory study., Methods: The right knees of 11 living subjects were scanned with a high-resolution computed tomography scanner at 0°, 30°, 60°, 90°, and 120° of knee flexion, and 3-dimensional (3D) models were constructed using customized software. Five patellar points were determined: 20% (point 20), 30% (point 30), 40% (point 40), 50% (point 50), and 60% (point 60) from the superior pole of the patella. The Schöttle femoral point (point F) was marked on a translucent 3D model of a true lateral view. Five virtual fibers connecting these points on the 3D knee model were created, and the lengths of various fibers were digitally measured., Results: The average length changes were 9.1±2.5 mm in F20, 9.1±2.5 mm in F30, 8.1±2.6 mm in F40, 6.9±2.4 mm in F50, and 6.9±1.7 mm in F60. There were significant differences in length changes of these 5 fibers (P<.001). The lengths of 2 superior fibers (F20 and F30) increased as the knee flexed from 0° to 30° and decreased as the knee flexed over 30°. The lengths of a middle fiber (F40) and an inferior fiber (F50) increased from 0° to 30°, reached a plateau from 30° to 60°, and then decreased from 60° to 120°. F60 showed an increase from 0° to 30°, and then a plateau pattern from 30° to 90°, followed by a decrease during further flexion., Conclusion: Superior fibers exhibited their maximum lengths at low flexion angles, and inferior fibers exhibited their maximum lengths at midflexion angles. The MPFL is a complex of functionally various fibers with some taut and others slack over the whole range of knee motion., Clinical Relevance: The results for lengths and length change patterns of various MPFL fibers are expected to serve as a theoretical background for anatomic double-bundle MPFL reconstruction., (© 2015 The Author(s).)