Age-related sarcopenia results in frailty and decreased mobility, which are associated with increased falls and long-term disability in the elderly. Given the global increase in lifespan, sarcopenia is a growing, unmet medical need. This report aims to systematically characterize muscle aging in preclinical models, which may facilitate the development of sarcopenia therapies. Naïve rats and mice were subjected to noninvasive micro X-ray computed tomography (micro-CT) imaging, terminal in situ muscle function characterizations, and ATPase-based myofiber analysis. We developed a Definiens (Parsippany, NJ)-based algorithm to automate micro-CT image analysis, which facilitates longitudinal in vivo muscle mass analysis. We report development and characterization of translational in situ skeletal muscle performance assay systems in rat and mouse. The systems incorporate a custom-designed animal assay stage, resulting in enhanced force measurement precision, and LabVIEW (National Instruments, Austin, TX)-based algorithms to support automated data acquisition and data analysis. We used ATPase-staining techniques for myofibers to characterize fiber subtypes and distribution. Major parameters contributing to muscle performance were identified using data mining and integration, enabled by Labmatrix (BioFortis, Columbia, MD). These technologies enabled the systemic and accurate monitoring of muscle aging from a large number of animals. The data indicated that longitudinal muscle cross-sectional area measurement effectively monitors change of muscle mass and function during aging. Furthermore, the data showed that muscle performance during aging is also modulated by myofiber remodeling factors, such as changes in myofiber distribution patterns and changes in fiber shape, which affect myofiber interaction. This in vivo muscle assay platform has been applied to support identification and validation of novel targets for the treatment of sarcopenia.