Skeletal muscle is the engine that powers what is arguably the most essential and defining feature of human and animal life—locomotion. Muscles function to change length and produce force to enable movement, posture, and balance. Despite this seemingly simple role, skeletal muscle displays a variety of phenomena that still remain poorly understood. These phenomena are complex—the result of interactions between active and passive machinery, as well as mechanical, chemical and electrical processes. The emergence of imaging technologies over the past several decades has led to considerable discoveries regarding how skeletal muscles function in vivo where activation levels are submaximal, and the length and velocity of contracting muscle fibres are transient. However, our knowledge of the mechanisms of muscle behaviour during everyday human movements remains far from complete. In this review, we discuss the principal advancements in imaging technology that have led to discoveries to improve our understanding of in vivo muscle function over the past 50 years. We highlight the knowledge that has emerged from the development and application of various techniques, including ultrasound imaging, magnetic resonance imaging, and elastography to characterise muscle design and mechanical properties. We emphasize that our inability to measure the forces produced by skeletal muscles still poses a significant challenge, and that future developments to accurately and reliably measure individual muscle forces will promote new frontiers in biomechanics, physiology, motor control, and robotics. Finally, we identify critical gaps in our knowledge and future challenges that we hope can be solved as a biomechanics community in the next 50 years.