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This study aimed to assess antioxidant effects of melatonin treatment compared to N-acetylcysteine (NAC) and to their combination in a sickle cell suspension. Sickle erythrocytes were suspended in phosphate-buffered saline, pH 7.4, composing external control group. They were also suspended and incubated at 37°C either in the absence (experimental control group) or in the presence of NAC, melatonin and their combination at concentrations of 100 pm, 100 nm and 100 μm for 1 hr (treatment groups). The melatonin influences were evaluated by spectrophotometric [hemolysis degree, catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), and superoxide dismutase (SOD) activities] and chromatographic methods [glutathione (GSH) and malondialdehyde (MDA) levels]. Incubation period was able to cause a rise about 64% on hemolysis degree as well as practically doubled the lipid peroxidation levels (P < 0.01). However, almost all antioxidants tested treatments neutralized this incubation effect observed in MDA levels. Among the antioxidant biomarkers evaluated, we observed a modulating effect of combined treatment on GPx and SOD activities (P < 0.01), which showed ˜25% decrease in their activities. In addition, we found an antioxidant dose-dependent effect for melatonin on lipid peroxidation (r = −0.29; P = 0.03) and for combined antioxidant treatments also on MDA levels (r = −0.37; P = 0.01) and on SOD activity (r = −0.54; P < 0.01). Hence, these findings contribute with important insight that melatonin individually or in combination with NAC may be useful for sickle cell anemia management.