•Evaluation of mitochondrial function in PBMC at different time points.•Sex differences in mitochondrial function and serum melatonin concentrations.•Mitochondrial parameters and melatonin blood levels were lower in males.•Endogenous melatonin concentration may influence mitochondrial function.•Studies on mitochondrial function should consider endogenous melatonin levels. Melatonin blood levels vary depending on the circadian rhythm. It also stimulates antioxidant enzymes and has positive effects on mitochondrial function. The current study investigated the effects of endogenously released melatonin on adenosine triphosphate (ATP) levels and mitochondrial respiration in peripheral blood mononuclear cells (PBMC). The current study included 20 healthy adults (mean age 25,7 ± 3.4 years). Blood was collected at 8 a.m. and 2 p.m. The activity of mitochondrial respiratory complexes and ATP levels were determined in isolated PBMC. Melatonin concentrations were determined in serum samples. Sleep behavior was assessed. In PBMCs isolated from blood samples of males, respiration of mitochondrial complex IV and ATP levels as well as serum melatonin concentration were significantly lower at 2 a.m. compared to the samples collected at 8 p.m. Mitochondrial parameters and melatonin blood levels were equal at both time points in the samples isolated from females. Although our results show that the amount of melatonin secreted may have had an influence, further investigation is needed to determine the importance of melatonin and other factors in measuring the mitochondrial function of PBMC.