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An experimental validation of an inversion procedure for retrieving the optical properties of layered media from multidistance time-resolved reflectance measurements is presented. The results cover a wide range of optical properties, showing excellent effectiveness and reliability of the procedure in reconstructing the optical properties of a two-layered medium. The optical properties of the first layer and the absorption of the second layer could be retrieved with excellent precision, whereas the reduced scattering coefficient of the second layer was reconstructed with a large error. The first layer thickness could be retrieved with an error less than 50%. An example of retrieval of the optical properties of muscle from in vivo measurements during an arterial occlusion is shown.