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In this randomized, double-blind human study, the short-term photoprotective effects of different antioxidants and their combinations were evaluated in vivo. Vitamin C (ascorbic acid), vitamin E (α-tocopherol) and melatonin (N-acetyl-5-methoxytryptamine) were topically applied, alone or in combination, 30 min before ultraviolet-irradiation of the skin. The erythemal reaction was evaluated visually and non-invasively using different bioengineering methods (skin colour and skin blood flow). The results showed a modest protective effect of the vitamins when applied alone and a dose-dependent photoprotective effect of melatonin. Topical application of combinations of both vitamins, or of melatonin with vitamins, enhanced the photoprotective response. Better protection was obtained by using the combination of melatonin with both vitamins. The role of reactive oxygen species and oxygen-derived free radicals, as well as potential sunscreening properties of the employed antioxidants, are discussed in view of possible mechanisms to explain this elevated photoprotective effect.