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The aim of this study was to investigate whether simultaneous irradiation at 660 and 808 nm generates different patterns of oxidative/antioxidative activities compared to consecutive irradiation. Primary cultures of gingival keratinocytes and fibroblasts were exposed to a diose laser (660 ± 2 nm and 808 ± 2 nm, 100 mW, 0.09 cm 2 spot area) using double irradiation with the two wavelengths (consecutive or simultaneous) for 6, 10, and 20 s. The two irradiation regimens did not increase cell viability in any of the experimental conditions. Lipid peroxidation was increased after consecutive irradiation in epithelial cells, which was not detected after simultaneous irradiation. After 20s of the simultaneous mode, ROS levels increased, but antioxidative balance decreased. In the fibroblasts, the two double irradiations induced ROS reduction, increase in lipid peroxidation, and improvement of antioxidative balance, mainly after the 20 s irradiation time. In conclusion, simultaneous and consecutive irradiation induced distinct oxidative stress modulation in oral epithelial cells and fibroblasts. The imbalance in the oxidative system observed after longer exposures, allied with the absence of a significant increase in the viability of the two cell types, suggests a contraindication for longer simultaneous irradiation in clinical situations that demand cellular stimulation., (© 2023 The American Society for Photobiology.)