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Abstract The knock-out mutation of the unique M13 family member, the Drosophila melanogaster (fruit fly) Neprilysin-like 15 ( Nepl15 ), resulted in marked reductions of glycogen and glycerolipid storage in adult male flies, but a significant increase of glycogen storage in adult female flies, although the mutant flies consumed the same amount of food as the isogenic w 1118 controls. The findings prompted us to characterize sex and age-specific effects of Nepl15 knock-out ( Nepl15 KO ) mutation on lifespan, fertility and fecundity, physiology, cytophysiology, and overall health. The current study shows Nepl15 transcripts are expressed in all embryonic stages of the control flies. The mutant embryos show more glycogen storage, likely due to more maternal glycogen deposition in the eggs. Moreover, there are slight increases in the number of eggs laid, the percentage of pupariation, and the percentage of adult fly eclosion from pupae in the Nepl15 KO mutant flies. Interestingly, Nepl15 KO female, but not male flies, outlive the respective control flies when cultured on a standard diet. The mutant adult females show significantly less Target of Rapamycin ( TOR ) and more Sirtuin 6 ( Sirt6 ) expression, changes that may synergistically contribute to their lifespan extension. In contrast, mutant males exhibit significant reductions in both TOR and Sirt6 expression, potentially offsetting their effects on longevity. Cellular health is further improved in mutant females, as evidenced by a marked reduction in reactive oxygen species (ROS), associated with a 1.5-fold increase in the Superoxide dismutase 2 ( Sod2 ) expression at 7 days of age. Both sexes demonstrate improved gut barrier integrity at 40 days, with reduced “Smurf” leakage compared to age-matched controls. Optical cardiography reveals that heart rate in 40-day-old mutants is better preserved, resembling that of 7-day-old flies, whereas control flies show a pronounced age-associated decline. Functionally, Nepl15 KO males and females outperform controls in a 6-cm climbing assay at 10, 20, 30, and 40 days of age, with the greatest difference observed at day 40. Following a 45-minute exercise bout at 10 rpm, mutant females continue to outperform controls at both 7 and 40 days, indicating preserved neuromuscular performance. Consistently, ATP levels are significantly elevated in 7- and 40-day-old mutant females, but not in mutant males. Interestingly, only 7-day-old mutant males exhibit increased mitochondrial inner membrane potential, which may enable more rapid ATP turnover when energy demand arises. No detectable differences are observed in thoracic muscle or mitochondrial ultrastructure, nor in overall mitochondrial number. However, no observable changes are noticed in the ultrastructure of the thoracic muscle and mitochondria, and the overall number of mitochondria in the mutant flies. Collectively, our findings demonstrate that Nepl15 loss-of-function confers health benefits at cellular, organ, and organismal levels, with pronounced sex-specific differences. However, the mechanisms by which aging mutant males sustain enhanced functional performance remain elusive.