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The proton transport properties of Nafion membranes were studied in a wide range of temperature by using an air-tight sample holder able to maintain the sample hydrated at high relative humidity. The proton conductivity of hydrated Nafion membranes continuously increased in the temperature range of 40–180 °C with relative humidity kept at RH = 100%. In the temperature range of 40–90 °C, the proton conductivity followed the Arrhenius-like thermal dependence. The calculated apparent activation energy Ea values are in good agreement with proton transport via the structural diffusion in absorbed water. However, at higher measuring temperatures an upturn of the electrical conductivity was observed to be dependent on the thermal history of the sample.