Cycle-dependent mechanical behavior of individual muscles in soft-bodied organisms revealed by scanning probe microscopy.

Soft-bodied organisms offer a fertile ground for uncovering fundamental principles of locomotion, yet direct mechanical access to their microscale muscles has remained elusive due to their fragility, compliance, and limited experimental accessibility. Here, we introduce a single-axis in situ tensile framework based on scanning probe microscopy (SPM), enabling direct cyclic stress-stretch measurements of individual muscles in Drosophila larvae with high spatial and force resolution. We reveal that these microscale muscles exhibit a distinctive cycle-dependent mechanical response, characterized by evolving J-shaped nonlinearity, progressive hysteresis modulation, and stiffness adaptation during repeated loading. Remarkably, this behavior is quantitatively captured by a minimal pseudo-elastic model, which links the observed hysteresis evolution to energy dissipation and potential structural adaptation at the sarcomeric scale. These findings provide new insights into the intrinsic passive mechanics of soft-bodied locomotion and establish a high-resolution experimental framework for investigating the mechanical behavior of biological soft tissues.