The egg vitelline membrane (VM) is a protein-based structure surrounding the yolk. It contributes to protect the egg from bacterial contaminations, and it enables the separation of white and yolk after egg breaking. Various factors, such as egg storage conditions and the age of laying hens, can affect the quality of this membrane. Considering the lengthening of the production period for laying hens, the aim of this study was to investigate the effects of both hen age and storage conditions on the physical and biochemical properties of the VM. Eggs from hens aged 32-34 (peak of lay), 71-73 (late laying period) and 94-99 (very late laying period) weeks were stored for up to 28 days at 4°C or 20°C. Several egg quality parameters were measured (n≥20 eggs per condition): yolk mass, yolk index, deformation and breaking strength of the VM. In parallel, VM samples were collected to analyze their protein composition by SDS-PAGE and mass spectrometry (6 pools of 4 egg samples per condition). As expected, the four quality parameters studied were significantly altered by both hen age and storage time. In particular, VM deformation and rupture strength in eggs produced by 94-99 week-old hens decreased by about 6% and 11-13%, respectively, compared with younger hens. A higher diminution of these two parameters was observed with storage time (average decrease of 24% and 28% in eggs stored for 28 days), regardless of hen age. High storage temperature also had a significant negative effect on these parameters, with the exception of yolk mass, which did not change significantly between 4°C and 20°C. Importantly, no interaction between hen age and storage conditions was observed in our conditions. The protein profile of solubilized VM was also altered, especially in eggs stored for 28 days at 20°C. At least 219 proteins were identified by the proteomic approach, and the quantification revealed differential relative abundances (Log2 Fold Change ≥ 1 or ≤ -1 with p-value ≤ 0.05) for half of these proteins as affected by hen age and/or storage conditions. In conclusion, increasing the production period of laying hens has a negative impact on the mechanical properties of the VM, but it does not seem to worsen its degradation kinetics during egg storage. In addition, our proteomic analyses allowed the identification of proteins potentially involved in alterations of the VM induced by hen age and egg storage.