Morning vs. afternoon saliva: limited diurnal variation in ion composition of unstimulated whole saliva

Saliva is increasingly recognized as a valuable diagnostic fluid due to its non-invasive collection and wide range of biochemical markers. However, the potential influence of the circadian rhythm on salivary composition remains a concern, e.g. in clinical diagnostics. This study aimed to determine whether the time of collection—morning (9:00–11:00) versus afternoon (13:00–15:00)—significantly affects ion concentrations in unstimulated whole saliva (UWS). Saliva samples from 30 healthy adults were analyzed for flow rate. Next, cortisol (as a reference marker for the circadian rhythm) and nine salivary ions were quantified using ELISA and capillary electrophoresis, respectively. No significant differences were found in flow rate or cortisol levels between the two timepoints. Most ion concentrations, namely sodium, potassium, chloride, phosphate, sulphate, ammonium, and nitrate, also remained stable. In contrast, calcium and magnesium showed significant, opposing shifts: calcium was higher in the afternoon, while magnesium was higher in the morning. These findings suggest that, with some exceptions, salivary ion concentrations are largely unaffected by collection time. This supports the flexible use of UWS for diagnostic purposes without strict timing constraints, increasing its applicability in clinical and research settings.