Environmental influences, especially temperatures below the freezing point, can affect the performance and long-term stability of PEMFCs. Within the scope of this research, a completely new test procedure was developed to characterize PEMFC single cells with respect to their long-term stability at temperature cycles between 80 °C and −10 °C. Using this procedure, the behavior of PEMFC single cells (active surface area of 43.6 cm2) with different cathode-ionomer-to-carbon (I/C) weight ratios (0.5/1.0/1.5) was evaluated. The generated in-situ measurement data clearly demonstrate that the performance of each PEMFC single cell changes individually as a function of the cathode I/C-ratio during the 120 stress cycles. While the MEA with an I/C ratio of 0.5 showed a power loss of ~1.49%, the MEAs with an I/C ratio of 1.0 and 1.5 showed a power loss of about ~7.75% and ~24.7%, respectively. The subsequent post-mortem ex-situ analyses clearly showed how the test procedure and the different I/C-ratios affected the changes in the catalyst layers (CL). The destructive mechanisms responsible for the changes can be divided into two categories: One part was driven by rapid enthalpy change leading to mechanical failure, and the other part, which led to the reduction of cathode CL thickness, was driven by rapid potential changes and potential shifts (overpotentials). This reduction in cathode CL thickness ultimately leads to an accumulation and excessive load of ionomer in the direction of GDL, resulting in a reduction in pore size, a shift in the core reaction area, and high O2 transport resistance.