OBJECTIVE To compare single-site intraoperative sarcomere length values with sarcomere lengths measured from systematic sampling of the entire transferred muscle. METHODS The tendon of the rabbit second toe extensor muscle was transposed to the ankle extensor retinaculum under levels of stretch over the sarcomere length range of about 2.5 microm to about 4.0 microm. Intraoperative sarcomere length was measured at a single site with a laser diffraction device. Whole-muscle sarcomere length measurement was then determined by sampling across the muscle in the proximal, middle, and distal regions. Linear regression analysis and intraclass correlation coefficients were used to validate single intraoperative sarcomere lengths relative to whole-muscle sarcomere lengths. RESULTS Single intraoperative sarcomere lengths correlated strongly with average whole-muscle sarcomere length, although there was a systematic tendency to overestimate intraoperative sarcomere length. Intraoperative sarcomere length also matched well with all regions sampled, indicating that there was no tendency for intraoperative sarcomere length to better represent one region of the muscle compared with another. CONCLUSIONS These results show that intraoperative sarcomere lengths accurately represent the entire muscle. The relatively small sarcomere length variations validate the use of intraoperative sarcomere length measurement during tendon transfer in which the entire muscle is not available for measurement because of limited surgical exposure.