To improve mass transport limitations in proton exchange membrane fuel cells (PEMFCs), a detailed analysis of the gas diffusion layer structure can aid to understand the transport processes of oxygen to the catalyst layer and the liquid water removal mechanism. Herein, the effect of tortuosity was studied using two different microporous layer (MPL) structures and their hydrophobic and hydrophilic clones. Focused ion beam-scanning electron microscopy tomography showed that all structures had similar porosity but different characteristic pore diameters. Volumetric analysis using the Laplace solver TauFactor and the PuMA random walk method yielded a tortuosity of 1.2-1.3 for both MPLs, and these values were confirmed by experiments based on limiting current measurements in a single-cell PEMFC. The inclusion of Knudsen diffusion in the MPL proved to be essential for both methods. Subsequent analysis using experimental operating conditions that induce liquid water formation suggested that different water filling mechanisms occur in MPLs with different surface wettability. A mechanistic understanding of the water filling processes was investigated by simulations, considering the effect of liquid water saturation in different pore size domains. It was found that the tortuosity increases more drastically when narrow pores are blocked by liquid water, consistent with the experiments.