Objectives: The study aimed to evaluate the impact of aging on the formation of neutrophil extracellular traps (NETs). The impaired formation of NETs is the cause of an abnormal innate immune response. Material and Methods: The study included a total of 45 healthy male subjects of different age groups. Whole blood was collected from the subjects, and the concentration of myeloperoxidase (MPO), the main biocidal protein in NETs, was determined in serum using ELISA. The serum levels of circulating free DNA (cfDNA), which are the structural basis of NETs, were also measured by fluorescence. In addition, the white blood cell count was determined, whole blood smear was evaluated, and the neutrophillymphocyte ratio was calculated. The variations in the levels of NET biomarkers were analyzed in different age groups. Results: The low levels of MPO (243.70 ng/ml) and cfDNA (6.24 ng/100 μl) in boys indicated neutrophil insufficiency for NETosis in children. A progressive increase in the levels of MPO and cfDNA with age was observed among adolescents (420.91, p = 0.04; 13.55, p = 0.03, respectively), with the highest level noted in the healthy adult group (466.58, p = 0.01; 14.07, p = 0.01, respectively). The levels of the studied parameters were comparable in adolescents and young adults, which proved that the NETosis process was appropriate and suggested the attainment of neutrophil maturity for the release of NETs in adolescence. The levels of MPO and cfDNA were low in older men (225.46, p < 0.01; 5.19, p < 0.01, respectively) indicating impaired NET formation. Conclusions: Data on the generation of NETs in different age groups obtained in this study can allow a better understanding of the ontogenesis of the immune system in terms of the course of NETosis, and also indicate the need to support nonspecific responses in children and adults. Further research should be performed to determine the possibility of regulating the NETosis process. Int J Occup Med Environ Health. 2023;36(3):333–48