Vitamin D binding protein (DBP) concentration is known to influence the availability and bioactivity of vitamin D metabolites but its diurnal rhythm (DR), its inter-relationships with the DRs of vitamin D metabolites and its influence on free vitamin D metabolite concentrations are not well described. The DRs of plasma total 25(OH)D, total 1,25(OH)2D, DBP, albumin and calculated free 25(OH)D and free 1,25(OH)2D were measured in men and women aged 60-75 years and resident in the UK (n 30), Gambia (n 31) and China (n 30) with differences in lifestyle, dietary intake and vitamin D status. Blood samples were collected every 4h for 24h and DRs statistically analysed with Fourier regression. Gambians had significantly higher plasma concentrations of vitamin D metabolites and lower albumin concentration compared to the British and Chinese. Significant DRs were observed for all analytes and calculated free vitamin D metabolites (P<0.01). The pattern of DRs was similar between countries. The magnitude of the DRs of free 1,25(OH)2D was attenuated compared to that of total 1,25(OH)2D whereas it was not different between total and free 25(OH)D. Relationships between the DRs were generally weak. There was no phase shift between 1,25(OH)2D and DBP with the strongest cross correlation at 0h time lag (r=0.15, P=<0.001). In comparison, 25(OH)D correlated less well with DBP (1h time lag, r=0.07, P=0.12). These data demonstrate a relationship between the DRs of 1,25(OH)2D and DBP, possibly to maintain free 1,25(OH)2D concentrations. In contrast, the DRs of total and free 25(OH)D appeared to be less influenced by DBP, suggesting that DBP has comparatively less effect on 25(OH)D concentration and 25(OH)D availability. This work highlights the importance of standardisation in timing of sample collection particularly for the assessment of plasma protein concentrations.