Using a 3D rotational shear wave elasticity imaging (SWEI) setup, 3D shear wave data were acquired in the vastus lateralis of a healthy volunteer. The innate tilt between the transducer face and the muscle fibers results in the excitation of multiple shear wave modes, allowing for more complete characterization of muscle as an elastic, incompressible, transversely isotropic (ITI) material. The ability to measure both the shear vertical (SV) and shear horizontal (SH) wave speed allows for measurement of three independent parameters needed for full ITI material characterization: the longitudinal shear modulus μL , the transverse shear modulus μT , and the tensile anisotropy χE . Herein we develop and validate methodology to estimate these parameters and measure them in vivo, with μL = 5.77±1.00 kPa, μT = 1.93±0.41 kPa (giving shear anisotropy χμ = 2.11±0.92 ), and χE = 4.67±1.40 in a relaxed vastus lateralis muscle. We also demonstrate that 3D SWEI can be used to more accurately characterize muscle mechanical properties as compared to 2D SWEI.