This study tested the common assumption that skeletal muscle shortens uniformly in the direction of its fascicles during low-load contraction. Cine phase contrast magnetic resonance imaging was used to characterize shortening of the biceps brachii muscle in 12 subjects during repeated elbow flexion against 5 and 15% maximum voluntary contraction (MVC) loads. Mean shortening was relatively constant along the anterior boundary of the muscle and averaged 21% for both loading conditions. In contrast, mean shortening was nonuniform along the centerline of the muscle during active elbow flexion. Centerline shortening in the distal region of the biceps brachii (7.3% for 5% MVC and 3.7% for 15% MVC) was significantly less (P < 0.001) than shortening in the muscle midportion (26.3% for 5% MVC and 28.2% for 15% MVC). Nonuniform shortening along the centerline was likely due to the presence of an internal aponeurosis that spanned the distal third of the longitudinal axis of the biceps brachii. However, muscle shortening was also nonuniform proximal to the centerline aponeurosis. Because muscle fascicles follow the anterior contour and centerline of the biceps brachii, our results suggest that shortening is uniform along anterior muscle fascicles and nonuniform along centerline fascicles.