We have recently demonstrated that the mitral valve anterior leaflet (MVAL) exhibited minimal hysteresis, no strain rate sensitivity, stress relaxation but not creep (Grashow, 2006, Ann Biomed Eng., 34(2), pp. 315–325;Grashow, 2006, Ann Biomed. Eng., 34(10), pp. 1509–1518). However, the underlying structural basis for this unique quasi-elastic mechanical behavior is presently unknown. As collagen is the major structural component of the MVAL, we investigated the relation between collagen fibril kinematics (rotation and stretch) and tissue-level mechanical properties in the MVAL under biaxial loading using small angle X-ray scattering. A novel device was developed and utilized to perform simultaneous measurements of tissue level forces and strain under a planar biaxial loading state. Collagen fibril D-period strain and the fibrillar angular distribution were measured under equibiaxial tension, creep, and stress relaxation to a peak tension of . Results indicated that, under equibiaxial tension, collagen fibril straining did not initiate until the end of the nonlinear region of the tissue-level stress-strain curve. At higher tissue tension levels, increased linearly with increasing tension. Changes in the angular distribution of the collagen fibrils mainly occurred in the tissue toe region. Using , the tangent modulus of collagen fibrils was estimated to be , which was times higher than the tissue tensile tangent modulus of . In creep tests performed at equibiaxial tension for , both tissue strain and remained constant with no observable changes over the test length. In contrast, in stress relaxation tests performed for was found to rapidly decrease in the first followed by a slower decay rate for the remainder of the test. Using a single exponential model, the time constant for the reduction in collagen fibril strain was , which was smaller than the tissue-level stress relaxation time constants of 22.0 and in the circumferential and radial directions, respectively. Moreover, there was no change in the fibril angular distribution under both creep and stress relaxation over the test period. Our results suggest that (1) the MVAL collagen fibrils do not exhibit intrinsic viscoelastic behavior, (2) tissue relaxation results from the removal of stress from the fibrils, possibly by a slipping mechanism modulated by noncollagenous components (e.g. proteoglycans), and (3) the lack of creep but the occurrence of stress relaxation suggests a “load-locking” behavior under maintained loading conditions. These unique mechanical characteristics are likely necessary for normal valvular function.