Objectives: Soft tissue exhibits nonlinear stress-strain behavior under compression. Characterizing its nonlinear elasticity may aid detection, diagnosis, and treatment of soft tissue abnormality. The purposes of this study were to develop a rate-controlled Mechano-Acoustic Indentor System and a corresponding finite element optimization method to extract nonlinear elastic parameters of soft tissue and evaluate its test-retest reliability. Methods: An indentor system using a linear actuator to drive a force-sensitive probe with a tip-mounted ultrasound transducer was developed. Twenty independent sites at the upper lateral quadrant of the buttock from 11 asymptomatic subjects (7 men and 4 women from a chiropractic college) were indented at 6% per second for 3 sessions, each consisting of 5 trials. Tissue thickness, force at 25% deformation, and area under the load-deformation curve from 0% to 25% deformation were calculated. Optimized hyperelastic parameters of the soft tissue were calculated with a finite element model using a first-order Ogden material model. Load-deformation response on a standardized block was then simulated, and the corresponding area and force parameters were calculated. Between-trials repeatability and test-retest reliability of each parameter were evaluated using coefficients of variation and intraclass correlation coefficients, respectively. Results: Load-deformation responses were highly reproducible under repeated measurements. Coefficients of variation of tissue thickness, area under the load-deformation curve from 0% to 25% deformation, and force at 25% deformation averaged 0.51%, 2.31%, and 2.23%, respectively. Intraclass correlation coefficients ranged between 0.959 and 0.999, indicating excellent test-retest reliability. Conclusions: The automated Mechano-Acoustic Indentor System and its corresponding optimization technique offers a viable technology to make in vivo measurement of the nonlinear elastic properties of soft tissue. This technology showed excellent between-trials repeatability and test-retest reliability with potential to quantify the effects of a wide variety of manual therapy techniques on the soft tissue elastic properties. ; Department of Health Technology and Informatics