The aging process is accompanied by an accumulation of cellular damage, which compromises the viability and function of cells and tissues. We aim to further explore the association between in vitro DNA damage markers and the chronological age of the donor, as well as long-lived family membership and presence of cardiovascular diseases. Therefore, numbers of 53BP1 foci, telomere-associated foci (TAF) and micronuclei were measured in cultured dermal fibroblasts obtained from three age groups of donors (mean age 22, 63 and 90 years). Fibroblasts were cultured without a stressor and with 0.6 μM rotenone for 3 days. We found that 53BP1 foci and TAF were more frequently present in fibroblasts of old donors compared to middle-aged and young donors. No association between micronuclei and donor age was found. Within the fibroblasts of the middle-aged donors we did not find associations between DNA damage markers and long-lived family membership or cardiovascular disease. Results were comparable when fibroblasts were stressed in vitro with rotenone. In conclusion, we found that DNA damage foci of cultured fibroblasts are significantly associated with the chronological age, but not biological age, of the donor.