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Background: This study aimed to examine the associations between physical fitness components and health-related quality of life (HRQoL) among adults stratified by sex and age. In addition, we aimed to examine whether these associations change based on socioeconomic, clinical, and biochemical characteristics. Methods: A total of 297 participants aged 47.41 (standard deviation: 9.08) years from the “Validity of a Model of Accelerated Vascular Aging as a Cardiovascular Risk Index in Healthy Adults: the EVasCu cross-sectional study” were included in this analysis. HRQoL, physical fitness, socioeconomic status (SES), waist circumference, and blood pressure were measured. Additionally, blood samples were extracted to determine cholesterol, triglyceride, and glycated hemoglobin A1c (HbA1c) levels. Analyses of covariance (ANCOVAs) were estimated to test mean differences in physical and mental health-related health measures (HRQoL) between fitness categories (fixed factors) by sex and age categories. Results: The physical HRQoL was related to the levels of fitness parameters among women, independent of age, while for men, it was related to better levels of general fitness and cardiorespiratory fitness among men aged < 50 and men aged ≥ 50, respectively. In contrast, mental HRQoL was related to cardiorespiratory fitness only among women aged < 50 years; speed/agility and flexibility among men aged < 50 years; and general fitness, strength, and flexibility among men aged ≥ 50 years. These data did not change when SES, clinical variables, or biochemical determinations were included in the analyses, neither for the physical nor for the mental HRQoL. Conclusion: Gender and age are important factors to be considered when analysing health indicators and influences in the population. In addition, SES, clinical characteristics, and biochemical parameters do not seem to influence the relationship between HRQoL and fitness.