Glucose metabolism marks health and disease and is causally inferred in the aging process. Ambulant continuous glucose monitoring provides 24-h glucose rhythms under daily life conditions. We aimed to describe ambulant 24-h glucose rhythms measured under daily life condition in relation to calendar and biological age in apparently healthy individuals. In the general population and families with propensity for longevity, we studied parameters from 24-h glucose rhythms; glucose levels; and its variability, obtained by continuous glucose monitoring. Participants were 21 young (aged 22–37 years), 37 middle-aged (aged 44–72 years) individuals from the general population, and 26 middle-aged (aged 52–74 years) individuals with propensity for longevity. All were free of diabetes. Compared with young individuals, middle-aged individuals from the general population had higher mean glucose levels (5.3 vs. 4.7 mmol L−1, P < 0.001), both diurnally (P < 0.001) and nocturnally (P = 0.002). Glucose variability was higher in the middle-aged compared with the young (standard deviation 0.70 vs. 0.57 mmol L−1, P = 0.025). Compared with middle-aged individuals from the general population, middle-aged individuals with propensity for longevity had lower overall mean glucose levels (5.2 vs. 5.4 mmol L−1, P = 0.047), which were more different nocturnally (4.8 vs. 5.2 mmol L−1, P = 0.003) than diurnally (5.3 vs. 5.5 mmol L−1, P = 0.14). There were no differences in glucose variability between these groups. Results were independent of body mass index. Among individuals without diabetes, we observed significantly different 24-h glucose rhythms depending on calendar and biological age.