DNAmPhenoAge, DNAmGrimAge, and the newly developed DNAmFitAge are DNA methylation (DNAm)-based biomarkers that reflect the individual aging process. Here, we examine the relationship between physical fitness and DNAm-based biomarkers in adults aged 33–88 with a wide range of physical fitness (including athletes with long-term training history). Higher levels of VO2max (ρ = 0.2, p = 6.4E − 4, r = 0.19, p = 1.2E − 3), Jumpmax (p = 0.11, p = 5.5E − 2, r = 0.13, p = 2.8E − 2), Gripmax (ρ = 0.17, p = 3.5E − 3, r = 0.16, p = 5.6E − 3), and HDL levels (ρ = 0.18, p = 1.95E − 3, r = 0.19, p = 1.1E − 3) are associated with better verbal short-term memory. In addition, verbal short-term memory is associated with decelerated aging assessed with the new DNAm biomarker FitAgeAcceleration (ρ: − 0.18, p = 0.0017). DNAmFitAge can distinguish high-fitness individuals from low/medium-fitness individuals better than existing DNAm biomarkers and estimates a younger biological age in the high-fit males and females (1.5 and 2.0 years younger, respectively). Our research shows that regular physical exercise contributes to observable physiological and methylation differences which are beneficial to the aging process. DNAmFitAge has now emerged as a new biological marker of quality of life.