To assess the intra-individual and inter-individuals biological variation and the effect of aging on lymphocyte T-cells subsets.We assessed lymphocyte phenotypes (CD3, CD4, and CD8 T-cells) in 89 HIV-1-infected and 88 uninfected white non-Hispanic men every 6 months, to examine the biological variation for those measurements, and the average change in lymphocyte phenotype over 34 years.The markers showed significant intra-individuality in HIV-infected and uninfected individuals with index of individuality of <1.4. No mean changes were seen over the 34 years, with the exception of percentage CD4T-cells in HIV-uninfected individuals.In the pre-HAART era, HIV-infected individuals experienced an increase in mean absolute CD3 T-cell numbers (11.21 cells/μL, P = 0.02) and absolute CD8 T-cell numbers (34.57 cell/μl, P < .001), and in the percentage of CD8 T-cells (1.45%, P < .001) per year and a significant decrease in mean absolute CD4 T-cell numbers (23.68 cells/μl, P < .001) and in the percentage of CD4 T-cells (1.49%, P < .001) per year.In the post-HAART era, no changes in mean levels were observed in absolute CD3 T-cell count (P = .15) or percentage (P = .99). Significant decreases were seen in mean count (8.56 cells/μl, P < .001) and percentage (0.59%, P < .001) of CD8 T-cells, and increases in mean absolute count (10.72 cells/μl, P < .001) and percentage (0.47%, P < .001) of CD4 T-cells.With the exception of CD4 (%), no average changes per year were seen in lymphocyte phenotype of HIV-uninfected men. The results of coefficients of variation of intra and inter-individuals of this study can be useful for HIV-1 infection monitoring and in addition the observation could be a useful guide for intra- and inter-individual coefficient variations, and establishing quality goal studies of different blood biomarkers in healthy and other diseases.