Micromechanical properties of single elastic fibers and fibrillin-microfibrils, isolated from equine ligamentum nuchae using chemical and enzymatic methods, were determined with atomic force microscopy (AFM). Young's moduli of single elastic fibers immersed in water, devoid of or containing fibrillin-microfibrils, were determined using bending tests. Bending freely suspended elastic fibers on a micro-channeled substrate by a tip-less AFM cantilever generated a force versus displacement curve from which Young's moduli were calculated. For single elastic fibers, Young's moduli in the range of 0.3-1.5 MPa were determined, values not significantly affected by the presence of fibrillin-microfibrils. To further understand the role of fibrillin-microfibrils in vertebrate elastic fibers, layers of fibrillin-microfibrils were subjected to nano-indentation tests. From the slope of the force versus indentation curves, Young's moduli ranging between 0.56 and 0.74 MPa were calculated. The results suggest that fibrillin-microfibrils are not essential for the mechanical properties of single vertebrate elastic fibers.