Diastolic dysfunction is a common phenotype across cardiovascular disease presentations. In addition to elevated cardiac stiffness (elevated left ventricular end-diastolic pressure), impaired cardiac relaxation is a key diagnostic indicator of diastolic dysfunction. While relaxation requires the removal of cytosolic calcium and deactivation of sarcomeric thin filaments, targeting such mechanisms has yet to provide effective treatments. Mechanical mechanisms, such as blood pressure (i.e., afterload), have been theorized to modify relaxation. Recently, we showed that modifying the strain rate of a stretch, not afterload, was both necessary and sufficient to modify the subsequent relaxation rate of myocardial tissue. The strain rate dependence of relaxation, called the mechanical control of relaxation (MCR), can be assessed using intact cardiac trabeculae. This protocol describes the preparation of a small animal model, experimental system and chamber, isolation of the heart and subsequent isolation of a trabecula, preparation of the experimental chamber, and experimental and analysis protocols. Evidence for lengthening strains in the intact heart suggests that MCR might provide new arenas for better characterization of pharmacological treatments, along with a method to assess myofilament kinetics in intact muscles. Therefore, studying the MCR may elucidate a path to novel approaches and new frontiers in the treatment of heart failure.