In a comparative study on erythrocytes (RBCs) drawn from mountaineers before and after a high-altitude stay, we observed that upon returning to sea level, their RBCs displayed a senescent-like phenotype as indicated by their density and the partial loss of membrane proteins which are shed by ageing RBCs. The aim of this study was to investigate possible changes in the membrane skeleton of these RBCs and to compare them with pathological RBCs. We analysed the proteins of RBC ghosts obtained from our subjects before and after returning to sea level by two-dimensional electrophoresis and mass spectrometry. We observed lower expression and fragmentation of β-actin after exposure to hypoxia. This suggested an alteration in membrane skeleton structure, which was confirmed by β-actin release in cell lysates during ghost preparation. We observed a similar actin fragmentation and release in RBC lysates from β-thalassaemic patients. In conclusion, these results indicate that after exposure to hypoxia, RBCs display a modification of their actin and cytoskeleton instability.