Integrated Opto–Biomechatronics For Single Muscle Fibre Structure‐Function Assessment: The <i>MyoRobot 3.0</i>

ABSTRACT Accurate assessment of biomechanical properties in single muscle fibres requires integrated structural and functional analysis. Conventional approaches assume constant fibre geometry during passive stretch and often use continuous stress–strain and stepwise stretch‐jump protocols interchangeably, despite their differing mechanical implications. We introduce the MyoRobot 3.0 , a next‐generation biomechatronics platform that integrates automated force measurements with fibre‐wide optical imaging. By synchronising a motorized optical system with controlled stretch velocities, the device maintains a constant field of view during elongation, enabling continuous diameter tracking along the same fibre segment. Using this approach, we quantified diameter thinning during axial stretch and compared slow continuous stress–strain recordings with rapid stepwise stretch‐jumps under varying calcium conditions and with or without cross‐bridge inhibition. Fibre diameter decreased by 13–15% at 40% strain, resulting in 27% cross‐sectional area loss and systematic underestimation of restoration stress when uncorrected. Continuous stress–strain protocols revealed calcium‐dependent increases in passive stiffness, whereas stepwise stretch‐jumps did not, indicating that the apparent calcium sensitivity partially reflects viscous or time‐dependent contributions. The MyoRobot 3.0 corrects major structural sources of error and enables protocol‐resolved biomechanical analysis, improving interpretation of passive muscle stiffness and its molecular determinants.