The material properties of the mitral valve chordae tendineae are important for the understanding of leaflet coaptation configuration and chordal pathology. This research combines basic histology with standard mechanical tests to determine the functioning role of the chordae tendineae during the cardiac cycle. Dual camera stereo photogrammetry was used to measure strains of the chordae in vitro under normal physiologic loading conditions. A uniaxial test simulating the same loading conditions was conducted. Histology and biochemical assays were performed on the chordae to determine chordal microstructure. Six porcine mitral valves were used for the in vitro flow loop study. The maximum strain experienced was 4.29% l 3.43% and was experienced at 249 msec after the start of valve closure. The loading rate was slightly higher than the unloading rate. The anterior lateral strut chordae had a higher maximum strain and loading rate than the posterior medial strut chordae. The posterior medial strut chordae had a higher unloading rate than the anterior lateral strut chordae. Histological examination revealed blood vessels in the chordae. The anterior strut chordae contain significantly more vessels than the other chordae. Different structural levels were observed for all chordae. The inner layer was characterized by a higher concentration of collagen; whereas, the middle layer was collagen with interwoven elastin fibers. The collagen microstructure was characterized by directional crimping. The anterior and posterior marginal chordae contained significantly more DNA than the other chordae (p<0.01). The anterior strut chord was found to contain significantly less DNA than all the other chordae (p<0.01). The collagen assay results showed that the posterior marginal chord contained significantly more collagen than the other chordae (p<0.01). The Fastin Elastin assay results showed no significant difference in the amount of elastin between the chordae. This study demonstrates the first in vitro examination of the strain experienced by the chordae tendineae of the mitral valve. This technique allows the investigation of the behavior of biological tissues under physiologic loading conditions. Contrary to earlier belief, vessels were found in the chordae. The microstructure and biochemical composition of the chordae tendineae is related to their function during coaptation.