Using a standardized immunophenotyping procedure we studied thirty-eight distinct subpopulations of T, B and NK lymphocytes in 253 healthy blood donors aged from 19 to 67. We analysed the influence of age, sex and HCMV seropositivity on each lymphocyte subpopulations and established reference ranges. We observed that aging influences the largest number of lymphocyte subpopulations with a slow increase of CD8+ EMRA T lymphocytes and of the numbers of circulating Tregs. The proportion of HLA-DR+ cells among Tregs increased with age and was correlated to the proportion of HLA-DR+ cells among effector T CD4+ lymphocytes. Sex had a major impact on absolute counts of CD4+ T cells which were higher in females. HCMV-seropositivity was associated with higher frequencies of CD8+ EMRA memory T lymphocytes while a high frequency of terminally differentiated EMRA CD4+ T cells was observed in 80% of HCMV-positive individuals and in none of the HCMV seronegative individuals.