Mechanical refining has potential application for overcoming lignocellulosic biomass recalcitrance to enzyme hydrolysis and improving biomass digestibility. This study highlighted the ability for a pilot scale disc refiner to improve the total carbohydrate conversion to sugars from 39% (unrefined hardwood sodium carbonate biomass) to 90% (0.13 mm gap, 20% consistency, ambient temperature) by optimizing the refining variables. The different biomass properties that changed with refining indicated the expected increase in sugar conversion. Controlling the refining parameters to narrower gaps and higher consistencies increased the resulting refined biomass hydrolysis. Positive correlations that increases in net specific energy (NSE) input and refining intensity (SEL) improved the enzymatic hydrolysis. In some severe cases, over-refining occurred when smaller gaps, higher consistencies, and more energy input reached a point of diminished return. The energy input in these scenarios, however, was much greater than realistically feasible for industrial application. Although well-established in the pulp and paper industry, gaps in understanding the fundamentals of refining remain. The observations and results herein provide the justification and opportunity for further mechanical refining optimization to maximize and adapt the mechanical refining technology for maximum efficiency within the process of biochemical conversion to sugar.