Over the past 50 years our understanding of the central role that muscle motion has in powering movement has accelerated significantly. Fundamental to this progress has been the development of methods for measuring the length of muscles and muscle fibers in vivo. A measurement of muscle fiber length might seem a trivial piece of information on its own. Yet when combined with knowledge of the properties of skeletal muscle it has proven a powerful tool for understanding the mechanics and energetics of locomotion and informing models of motor control. In this perspective we showcase the value of direct measurements of muscle fiber length from four different techniques:​ sonomicrometry, fluoromicrometry, magnetomicrometry, and ultrasound. For each method, we review its history and provide a high-level user’s guide for researchers choosing tools for measuring muscle length in vivo. We highlight key insights that these measurements have provided, including the importance of passive elastic mechanisms and how skeletal muscle properties govern locomotor performance. The diversity of locomotor behaviors revealed across comparative studies has provided an important tool for discovering the rules for muscle function that span vertebrate locomotion more broadly, including in humans.