The results on determination of glucose and glycerol diffusion coefficients in myocardium tissue are presented. The method is based on the measurement and analysis of temporal dependence of tissue optical collimated transmittance under action of a hyperosmotic agent. This temporal tissue response is related to the rate of the agent and water diffusion in a tissue. The diffusion coefficients for tissue fluid fluxes at glucose and glycerol application to the myocardium at 20℃ have been estimated as (4.75 ± 3.40) × 10⁷ and (7.71 ± 4.63) × 10⁷ cm²/s, respectively.