Aggregation in heat-treated Nafion ionomer dispersion and 117 membrane are investigated by 1H and 19F Nuclear Magnetic Resonance (NMR) spectra, spin-lattice relaxation time, and self-diffusion coefficient measurements. Results demonstrate that heat-treatment affects the average Nafion particle size in aqueous dispersions. Measurements on heat-treated Nafion 117 membrane show changes in the 1H isotropic chemical shift and no significant changes in ionic conductivity. Scanning electron microscopy (SEM) analysis of prepared cathode catalyst layer containing the heat-treated dispersions reveals that the surface of the electrode with the catalyst ink that has been pretreated at ca. 80 °C exhibits a compact and uniform morphology. The decrease of Nafion ionomer’s size results in better contact between catalyst particles and electrolyte, higher electrochemically active surface area, as well as significant improvement in the DMFC’s performance, as verified by electrochemical analysis and single cell evaluation.