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Photobiomodulation therapy (PBMT) causes stimulatory effects that raise cell metabolism. The study aimed to evaluate the effects of PBMT on the endothelial function of healthy individuals. It was a controlled, randomized, crossover, triple-blind trial with 22 healthy volunteers (female: 77.3%), aged 25.45 years which were randomly divided into three groups. PBMT with gallium-aluminum-arsenide (GaAlAs) diode laser (810 nm, continuous-wave mode, 1000 mW, 0.28 cm 2 ) was applied over the radial and ulnar artery regions in two parallel spots: group 1-30 J (n = 22, 107 J/cm 2 ) per spot; group 2-60 J (n = 22, 214 J/cm 2 ) per spot; and group 3-placebo (n = 22, sham). The endothelial function was measured before and immediately after PBMT by the flow-mediated dilation technique (%FMD) with high-resolution ultrasound. Statistical analysis was made with ANOVA for repeated measures, the effect size was measured by Cohen's d, and results are presented as mean and standard error (or 95% confidence intervals). A p-value < 0.05 was considered statistically significant. The %FMD increases 10.4% with 60 J (mean difference = 0.496 mm, 95% CI = 0.42 to 0.57, p < 0.001), 7.3% with 30 J (mean difference = 0.518 mm, 95% CI = 0.44 to 0.59, p < 0.001), and 4.7% with placebo (mean difference = 0.560 mm, 95% CI = 0.48 to 0.63, p < 0.001). We found a small effect size (p = 0.702; d de Cohen = 0.24) without statistical difference between interventions. PBMT with the energy density of 60 J and 30 J did not improve endothelial function.Trial registration number: NCT03252184 (01/09/2017)., (© 2023. The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature.)