Timed immersion in buffered ethylenediamine-tetraacetic acid (EDTA) was used to selectively alter the mineral content at each level in the cortical bone structural hierarchy. The effects on the mechanical behavior were investigated using a combination of experimental techniques which provide collectively a wide range of resolution (5 μm to 3mm). Optical microscopy and histological analysis demonstrated a heterogeneous structure consisting of a mineralized tissue core surrounded by a layer of demineralized tissue (collagen) whose thickness varied depending on the immersion time. The mechanical behaviors of treated samples with (intact) and without (core) the surrounding demineralized layer were evaluated using three-point flexure. Overall, the intact specimens became significantly less brittle with increased immersion time in buffered-EDTA. For the core specimens, there was a systematic decrease in the elastic flexural properties (E, σe, εe). The site-specific properties of the specimens were determined using microhardness testing, scanning acoustic microscopy, and wavelength dispersive analysis. The mineralization and site-specific properties of the mineralized cores were not significantly affected by buffered-EDTA immersion; however, histomorphometric analysis showed a decrease in the mineralized volume fraction via widening of the pre-existing vascular channels. The experimental hierarchy was effective in discerning site-specific property changes and the localized heterogeneities resulting from the buffered-EDTA treatment. Based on the results of this study, buffered-EDTA treatment can be used to facilitate the determination of material and physical properties of intact and demineralized tissues within a single cortical bone sample.