Assembly pressure plays an important role in the factors affecting the performance of a PEM fuel cell. An insufficient clamping pressure may cause large contact resistance and thus lower the cell performance. On the other hand, over-clamping may reduce the porosity and permeability of the gas diffusion layer (GDL) and also result in poor cell performance. Therefore, it is very important to determine the proper assembly pressure for obtaining optimal performance. In this study, we design a special test fixture to evaluate the effect of assembly pressure on the performance of a PEM fuel cell. Without disassembling the fuel cell, the clamping pressure can be adjusted in situ to measure the cell performance directly and precisely. The unique single cell design eliminates the influence of gasket around the membrane electrode assembly (MEA) and makes it possible to estimate the compression effect of GDL independently. Three different types of carbon paper are used in the experiments as the GDLs. The variations of water contact angle, gas permeability, and in-plane electrical resistivity with the assembly pressure are also measured to explore the effects of assembly pressure on these physical properties. The results show that an optimal assembly pressure is always observed in each case, indicating an adequate compression on GDL is quite necessary for fuel cells.