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We present a method to measure effective diffusion coefficients of fluorescently labeled molecules inside a nanofluidic system. Molecules with small diffusion coefficients show a larger lateral dispersion than highly diffusive species, which is counterintuitive. We performed measurements with wheat germ agglutinin proteins and obtained an effective diffusion coefficient which is four orders of magnitude lower than its free diffusion coefficient. Our technique which is a direct and relatively simple measurement of the effective diffusion coefficients inside nanochannels of well controlled dimensions could help fundamental studies in membranes and separation sciences.