When pretreating woody biomass for the production of cellulosic ethanol, a mechanical downsizing step is commonly included to ensure an appropriate particle size for enzyme hydrolysis. Different methods of mechanical downsizing will result in wood particles with markedly different physical structures. Dry grinding methods, such as knife-milling, will produce a powder-like substrate, which consists of cut or truncated fiber bundles. The substrate will also have a reduced pore volume because of the required drying. Using a disc-refiner, wet wood chips are separated into single wood fibers and loosened fiber bundles, increasing available surface area and avoiding pore collapse because of drying. The following study compared knife-milled and disc-refined substrates produced from native and dilute-acid-pretreated wood chips to determine the impact of the mechanical-downsizing method on the enzyme digestibility and physical characteristics of a hardwood substrate. For dilute-acid-pretreated aspen, disc-refining produced a substrate that was 58-80% digestible, while knife-milling produced a substrate that was 24-36% digestible. The difference in substrate digestibility was partially attributed to hornification during the drying step and also attributed to differences in physical structure because of the downsizing method. Analysis via microscopy indicated that disc-refined substrates had a greater length, smaller width, and greater fibrillation then the knife-milled substrates. The disc-refined substrates also had a more exposed cellulose surface and a greater volume of accessible pores.