Sarcomere length is a key physiological parameter that affects muscle force output; however, our understanding of the scaling of human muscle from sarcomere to whole muscle is based primarily on cadaveric data. The aims of this study were to explore the in vivo relationship between passive fascicle length and passive sarcomere length at different muscle-tendon unit lengths and determine whether sarcomere and fascicle length relationships are the same in different regions of muscle. A microendoscopy needle probe capable of in vivo sarcomere imaging was inserted into a proximal location of the human tibialis anterior muscle at three different ankle positions [5° dorsiflexion, 5° plantar flexion (PF), and 15° PF] and one distal location at a constant ankle position (5° PF distal). Ultrasound imaging of tibialis anterior fascicles, centered on the location of the needle probe, was performed for each condition to estimate fascicle length. Sarcomere length and fascicle length increased with increasing muscle-tendon unit length, although the correlation between sarcomere length change and muscle fascicle length change was only moderate (r2 = 0.45). Passive sarcomere length was longer at the distal imaging site than the proximal site (P = 0.01). When sarcomere number was estimated from sarcomere length and fascicle length, there were fewer sarcomeres in the fibers of distal location than the proximal location (P = 0.01). These data demonstrate that fascicle length changes are representative of sarcomere length changes, although significant variability in sarcomere length exists within a muscle and sarcomere number per fiber is region-dependent.NEW & NOTEWORTHY Sarcomere and fascicle lengths were measured in vivo from human muscle to examine the relationship between the different scales of organization. Changes in fascicle length were moderately related to sarcomere length changes; however, sarcomere length and number per fiber varied from proximal to distal regions of the muscle. Differences in average sarcomere operating lengths across the muscle suggest potentially different stresses or strains experienced within different regions of muscle.