Purpose Various strategies for improving reliability of fascicle identification on ultrasound images are used in practice, yet these strategies are untested for effectiveness. Studies suggest that the largest part of differences between fascicle lengths on one image are attributed to the error on the initial image. In this study, we compared reliability results between different strategies. Methods Static single-image recordings and image sequence recordings during passive ankle rotations of the medial gastrocnemius were collected. Images were tracked by three different raters. We compared results from uninformed fascicle identification (UFI) and results with information from dynamic length changes, or data-informed tracking (DIT). A second test compared tracking of image sequences of either fascicle shortening (initial-long condition) or fascicle lengthening (initial-short condition). Results Intra-class correlations (ICC) were higher for the DIT compared to the UFI, yet yielded similar standard error of measurement (SEM) values. Between the initial-long and initial-short conditions, similar ICC values, coefficients of multiple determination, mean squared errors, offset-corrected mean squared errors and fascicle length change values were found for the DIT, yet with higher SEM values and greater absolute fascicle length differences between raters on the first image in the initial-long condition and on the final image in the initial-short condition. Conclusions DIT improves reliability of fascicle length measurements, without lower SEM values. Fascicle length on the initial image has no effect on subsequent tracking results. Fascicles on ultrasound images should be identified by a single rater and care should be taken when comparing absolute fascicle lengths between studies.