Macrophage polarization is a candidate biomarker of disease-related inflammatory status, but its modulation during aging has not been investigated. To do this, the M1/M2 profile was assessed by CD80/CD163 gating in classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14lowCD16+) monocytes from 31 healthy subjects (CTRs) of different ages. Cytofluorimetric analysis showed a significantly different CD80/CD163 distribution in the three subsets, as more than 80% of classical and intermediate monocytes were CD80+CD163+, whereas most non-classical monocytes were CD80-CD163- and CD163+. Non-classical CD163+ monocytes were significantly higher whereas classical CD163+ and CD80-CD163- monocytes significantly lower in older than younger CTRs (cut-off, 65 years), suggesting different age-related trends for M2 subsets. To establish whether an M1/M2 imbalance could be associated with disease, 21 patients with acute myocardial infarction (AMI) were compared with older CTRs. The AMI patients showed a significantly decreased proportion of CD163+CD80+ and an increased proportion of CD163+ and CD163-CD80- cells among classical monocytes, opposite trends to those observed in healthy aging. Moreover, a significantly greater proportion of intermediate and non-classical CD80+ monocytes suggested a shift to a pro-inflammatory phenotype. Overall, CD163/CD80 cytofluorimetric characterization of circulating monocytes provides additional information about their polarization and could be an innovative tool to monitor aging.