Understanding the complex relationships between microstructural organization and macromechanical function is fundamental to our knowledge of the differences between normal, diseased/injured, and healing connective tissues. The long-term success of functional tissue-engineered constructs or scaffolds may largely depend on our understanding of the structural organization of the original tissue. Although innovative techniques have been used to characterize and measure the microstructural properties of collagen fibers, a large gap remains in our knowledge of the behavior of intermediate scale (i.e., “mesostructural”) groups of fiber bundles in larger tissue samples. The objective of this study was to develop a system capable of directly measuring deformations of these smaller mesostructures during application of controlled loads. A novel mesostructural testing system (MSTS) has been developed to apply controlled multiaxial loads to medium (meso-) scale tissue specimens, while directly measuring local nonuniform deformations using synchronized digital video capture and “markerless” image correlation. A novel component of the MSTS is the use of elliptically polarized light to enhance collagen fiber contrast, providing the necessary texture for accurate markerless feature tracking of local fiber deformations. In this report we describe the components of the system, its calibration and validation, and the results from two different tissues: the porcine aortic valve cusp and the bovine pericardium. Validation tests on prepared samples showed maximum error of direct strain measurement to be 0.3%. Aortic valve specimens were found to have larger inhomogeneous strains during tensile testing than bovine pericardium. Clamping effects were more pronounced for the valve specimens. A new system for direct internal strain measurement in connective tissues during application of controlled loads has been developed and validated. The results from the two different tissues show that significant inhomogeneous deformations can occur even in simple tensile testing experiments.