The objective of this work was to investigate the effect of cake collapse during freeze-drying on the stability of protein lyophilizates containing a monoclonal IgG(1)-antibody or a second pharmaceutically relevant protein, referred to as PA01. In addition, L-lactic dehydrogenase was investigated because of its well-documented sensitivity towards freeze-drying stresses. Collapse was induced by two different means. First, by varying the ratio of the crystalline bulking agent mannitol to the amorphous stabilizer sucrose, different extents of collapsed cakes were generated. Second, formulations were freeze-dried using an aggressive collapse-cycle and a conventional freeze-drying protocol and collapsed and noncollapsed cakes of identical formulation were produced. Lyophilizates were analyzed using a comprehensive set of analytical techniques to monitor protein stability in terms of formation of soluble and insoluble aggregates, the biological activity and the conformational stability. The stability of excipients, namely the glass transition temperature, crystallinity, reconstitution behavior, and the residual moisture content was analyzed as well. In addition, the extent of collapse was quantified using the decrease of the specific surface area (SSA). Collapsed cakes had comparable residual moisture levels to noncollapsed lyophilizates. Reconstitution times were not increased. Protein stability was not relevantly different between collapsed and noncollapsed cakes.