Muscle contractures that occur after upper motor neuron lesion are often surgically released or lengthened. However, surgical manipulation of muscle length changes a muscle's sarcomere length (Ls), which can affect force production. To predict effects of surgery, both macro- (fascicle length (Lf)) and micro- (Ls) level structural measurements are needed. Therefore, the purpose of this study was to quantify both Ls and Lf in patients with cerebral palsy (CP) as well as typically developing (TD) children. Soleus ultrasound images were obtained from children with CP and TD children. Lf was determined and, with the joint in the same position, CP biopsies were obtained and formalin fixed, and Ls was measured by laser diffraction. Since soleus Ls values were not measurable in TD children, TD Ls values were obtained using three independent methods. While average Lf did not differ between groups (CP = 3.6 ± 1.2 cm, TD = 3.5 ± 0.9 cm; p > 0.6), Ls was dramatically longer in children with CP (4.07 ± 0.45 μm vs. TD = 2.17 ± 0.24 μm; p < 0.0001). While Lf values were similar between children with CP and TD children, this was due to highly stretched sarcomeres within the soleus muscle. Surgical manipulation of muscle-tendon unit length will thus alter muscle sarcomere length and change force generating capacity of the muscle. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:33–39, 2015.