The relationship between muscle thickness and pennation angle is mediated by fascicle length in the muscles of the lower extremities.

Muscle morphological architecture, a crucial determinant of muscle function, has fascinated researchers since the Renaissance. Imaging techniques enable the assessment of parameters such as muscle thickness (MT), pennation angle (PA), and fascicle length (FL), which may vary with growth, sex, and physical activity. Despite known interrelationships, robust mathematical models like causal mediation analysis have not been extensively applied to large population samples. We recruited 109 males and females, measuring knee flexor and extensor, and plantar flexor MT, PA, and FL using real-time ultrasound imaging at rest. A mixed-effects model explored sex, leg (dominant vs. non-dominant), and muscle region differences. Males exhibited greater MT in all muscles (0.1 to 2.1 cm, p < 0.01), with no sex differences in FL. Dominant legs showed greater rectus femoris (RF) MT (0.1 cm, p = 0.01) and PA (1.5°, p = 0.01), while vastus lateralis (VL) had greater FL (1.2 cm, p < 0.001) and PA (0.6°, p = 0.02). Regional differences were observed in VL, RF, and biceps femoris long head (BFlh). Causal mediation analyses highlighted MT's influence on PA, mediated by FL. Moderated mediation occurred in BFlh, with FL differences. Gastrocnemius medialis and lateralis exhibited FL-mediated MT and PA relationships. This study unveils the intricate interplay of MT, FL, and PA in muscle architecture.