Emphysema is a debilitating disease that remodels the lung leading to reduced tissue stiffness. Thus, understanding emphysema progression requires assessing lung stiffness at both the tissue and alveolar scales. Here, we introduce an approach to determine multiscale tissue stiffness and apply it to precision-cut lung slices (PCLS). First, we established a framework for measuring stiffness of thin, disk-like samples. We then designed a device to verify this concept and validated its measuring capabilities using known samples. Next, we compared healthy and emphysematous human PCLS and found that the latter was 50% softer. Through computational network modeling, we discovered that this reduced macroscopic tissue stiffness was due to both microscopic septal wall remodeling and structural deterioration. Lastly, through protein expression profiling, we identified a wide spectrum of enzymes that can drive septal wall remodeling, which, together with mechanical forces, lead to rupture and structural deterioration of the emphysematous lung parenchyma.