A thorough understanding of thermal effects on the physicochemical properties of layer-by-layer (LbL) assembled multilayers is crucial in utilizing these films in a variety of applications. In this work, we investigate the effect of thermal treatment on cross-linking and swelling of a hydrogen-bonded multilayer film made of poly(acrylic acid) (PAA) and polyacrylamide (PAAm), which has been shown to exhibit excellent long-term cell adhesion resistance. We observe that the apparent swelling of PAA/PAAm multilayers treated at 90 and 180 degrees C in a physiologically relevant condition is similar; however, these two multilayers with different thermal history exhibit completely different cell adhesion properties when assessed with human mesenchymal stem cells (hMSCs). While the 90 degrees C treated samples show excellent cell adhesion resistance, those treated at 180 degrees C are highly cell adhesive. A combination of characterization techniques including thermogravimetric analysis (TGA) and Fourier-transform infrared (FT-IR) spectroscopy reveals that complete cross-linking between PAA and PAAm chains occurs above 150 degrees C. PAA/PAAm multilayer films incubated at temperatures below 150 degrees C have a very low degree of cross-linking. Thus, when these less-cross-linked films are exposed to aqueous solutions of pH 4 or higher, a significant loss of the constituent materials is observed. This lightly cross-linked hydrogel-like network in phosphate buffered saline (PBS) has a water content of similar to 94 vol %, making the surface cell-adhesion resistant. In constrast, PAA/PAAm films incubated at 180 degrees C consist of similar to 72 vol % water in PBS and exhibit cell-adhesive properties. The shear modulus of 180 degrees C treated films measured by quartz crystal microbalance with dissipation (QCM-D) monitoring is 6.0 MPa, which is 2 orders of magnitude higher than that of lightly cross-linked 90 degrees C treated films (0.02 MPa), suggesting that mechanical compliance plays a significant role in influencing the cell adhesion behaviors. This work emphasizes the importance of thermal treatment conditions, which potentially can be used as a postassembly route to gradually modify and control the properties of LbL multilayers over a wide range for specific applications.