Recent evidence suggests that dietary intake of vitamins, in particular the B-vitamins including B6, B9 and B12 may have a number of positive effects on mood and stress. Given the effects of stress on a range of biological mechanisms including the endocrine system, it could be reasonably expected that multivitamin supplementation may also affect markers of these mechanisms such as diurnal cortisol secretion. In the current double-blind placebo-controlled study 138 adults (aged 20 to 50 years) were administered a multivitamin containing B-vitamins versus placebo over a 16-week period. Salivary cortisol measurements were taken at waking, 15-min, 30-min and at bedtime, at baseline, 8-weeks and 16-weeks. Perceived Stress (PSS) was measured at baseline, 8-weeks and 16-weeks, while blood serum measures of B6, B12 and homocysteine (HCy) as well as red cell folate (B9) were also collected at these time points. A significant interaction was found between treatment group and study visit for the Cortisol Awakening Response (CAR). Compared to placebo, at 16-weeks multivitamin supplementation was found to be associated with a near-significant trend towards an increased CAR. No significant differences in PSS were found between groups, with PSS increasing in both groups across the course of the study. Red cell folate was found to be significantly correlated with the CAR response at 16-weeks while HCy levels were not found to be associated with the CAR response, although HCy significantly correlated with waking cortisol levels at 8-weeks. A possible interpretation of the elevation in CAR associated with multivitamin supplementation is that this represents an adaptive response to everyday demands in healthy participants.