Gas diffusions layers (GDLs) constitute vital subcomponents of proton exchange membrane fuel cells (PEMFCs) since they participate in all relevant transport processes (gases, vapor, liquid water, electricity and heat).Meanwhile, state-of-the-art GDLs are produced on commercial scale using high volume reel-to-reel (R2R) processes. In almost any case, GDLs are designed as dual-layer structures with a macro-porous carbon fiber-based substrate (backing) and a micro-porous layer (MPL) consisting of carbon particulates bonded with polytetrafluoroethylene (PTFE) binder. A body of scientific work has verified that different aspects of the MPL microstructure and the MPL/macroporous substrate interface play a substantial role in the water management of PEMFCs [1-6].A systematic study was performed based on a singular backing using different MPL types and penetration levels.A simple method to estimate the micro-porous layer penetration in gas diffusion layers based on data obtained from force-deflection behavior of backings and MPL-coated GDLs is proposed. Comparison with SEM and previously published tomographic studies shows that a good estimate for the extent of MPL intrusion into the backing is obtained.MPL loading and penetration is shown to affect different ex-situ GDL properties (in-plane gas permeability, electrical and thermal conductivity) as well as the GDL single cell performance characteristics with respect to humification and high current density operation.Keywords: Gas diffusion layers, Microporous Layer, PEMFC fuel cells Weber, and J. Newman, “Effects of microporous layers in polymer electrolyte fuel cells”, J. Electrochem. Soc. 152, A677-A688 (2005).Lee, R. Yip, P. Antonacci, N. Ge, T. Kotaka, Y. Tabuchi, and A. Bazylak, Synchrotron investigation of microporous layer thickness on liquid water distribution in a PEM fuel cell, J. Electrochem. Soc. 162, F669-F676 (2015).Zhou, S. Shukla, A. Putz, and M. Secanell, “Analysis of the role of the microporous layer in improving polymer electrolyte fuel cell performance”, Electrochim. Acta. 268, 366-382 (2018).Cho, J. Park, H. Oh, K. Min, E. Lee, and J.Y. Jyoung, “Analysis of the transient response and durability characteristics of a proton exchange membrane fuel cell with different micro-porous layer penetration thicknesses”, Appl. Energy 111, 300-309 (2013).T. Gostick, M. A. Ioannidis, M. W. Fowler, and M. D. Pritzker, “On the role of the microporous layer in PEMFC operation.”, Electrochem. Comm. 11, 576-579 (2009).P. Ramasamy, E. C. Kumbur, M. M. Mench, W. Liu, D. Moore, and M. Murthy, “Investigation of macro- and micro-porous layer interaction in polymer electrolyte fuel cells”, Int. J. Hydrogen Energy 33, 3351-3367 (2008).