Particle lithography is a relatively simple, inexpensive technique used to pattern inorganics, metals, polymers, and biological molecules on the micro- and nanometer scales. Previously, we used particle lithography to create hexagonal patterns of protein dots in a protein resistant background of methoxy-poly(ethylene glycol)-silane (mPEG-sil). In this work, we describe a simple heating procedure to overcome a potential limitation of particle lithography: the simultaneous change in feature size and center-to-center spacing as the diameter of the spheres used in the lithographic mask is changed. Uniform heating was used to make single-diameter protein patterns with dot sizes of approximately 2-4 or 2-8 μm, depending on the diameter of the spheres used in the lithographic mask, while differential heating was used to make a continuous gradient of dot sizes of approximately 1-9 μm on a single surface. We demonstrate the applicability of these substrates by observing the differences in neutrophil spreading on patterned and unpatterned protein coated surfaces.