Abstract

Saliva serves as a valuable, non-invasive diagnostic tool, yet the influence of circadian rhythms on its biochemical composition remains unclear. This study evaluated circadian fluctuations in unstimulated whole saliva, focusing on pH, total protein, glucose, and organic and inorganic ions. Eight healthy, caries-free adults provided unstimulated saliva samples at six time points (7:00 AM, 11:00 AM, 3:00 PM, 7:00 PM, 11:00 PM, and 3:00 AM) across a 24-hour period, on three separate days. Participants followed a standardized Mediterranean diet for three days before and during sampling. Saliva was analyzed for pH, total protein (bicinchoninic acid assay), and various biochemical markers using a calibrated reflectometer. Statistical analyses were performed using repeated measures ANOVA (p <0.05). No significant circadian variations were observed for calcium, phosphate, peroxide, salivary pH, or total protein (p >0.05). However, lactate, nitrate, nitrite, ammonium, and glucose exhibited significant circadian fluctuations (p <0.05), with distinct peaks at specific times. Circadian rhythm had a limited impact on the overall biochemical profile of unstimulated saliva in young, healthy adults. However, specific analytes exhibited significant fluctuations, highlighting the need for standardized sampling protocols in saliva-based diagnostics. Understanding circadian influences on saliva composition may improve the reliability of saliva as a diagnostic biofluid for oral and systemic conditions. © 2025 Taylor & Francis Group, LLC.