Abstract.BACKGROUND: Salivary markers of oxidative stress and antioxidant status represent promising tool for the research of oral diseases. One of the criteria is the validation of these biomarkers from the perspective of the confounding and modifying factors. AIM: To examine the effect of circadian rhythm, tooth-brushing and ascorbic acid treatment on selected salivary markers of oxidative and carbonyl stress, and antioxidant status. SUBJECTS AND METHODS: Whole unstimulated saliva samples were collected from 19 healthy participants three times during a day, before and after tooth-brushing, and before and after the administration of vitamin C (250 mg). Advanced oxidation protein products (AOPP), thiobarbituric acid reactive substances (TBARS), advanced glycation end products (AGEs), ferric reducing antioxidant power (FRAP) and total antioxidant capacity (TAC) were measured. RESULTS: Salivary AGEs levels varied significantly during the day (p < 0.05) with the highest concentrations in the morning. FRAP levels varied during the day (p < 0.01) with the highest concentrations in the afternoon. Tooth-brushing decreased AGEs (p < 0.05) and TBARS levels (p < 0.01) and increased FRAP levels (p < 0.05). Single intake of vitamin C significantly decreased AGEs (p < 0.001) and increased both FRAP (p < 0.01) and TAC (p < 0.01) concentrations. CONCLUSION: Significant daily variations were observed in salivary AGEs and FRAP levels. Tooth-brushing and treatment with vitamin C decreased carbonyl stress and increased the antioxidant status. These results are important from the perspective of using saliva for the research of oral diseases.