Abstract Background Vitamin D nutrition research requires accurate measures of circulating 25-hydroxyvitamin D. Our objectives were to test whether a diurnal fluctuation in blood-spot concentrations of 25-hydroxyvitamin D can be demonstrated statistically in a single individual, and whether such fluctuation is affected by the pre-dose versus post-dose timing of the blood draw. Case presentation The participant in this case study was a generally healthy Caucasian woman in her 40s who has taken 5000 IU vitamin D3 supplement at midday for over 1 year. Each blood sample was drawn individually from a finger prick onto filter paper at morning, midday, or night, on 4 days (three groups of five individual blood samples per collection day). On days 1 and 2, the midday samples were collected approximately 1 hour after the supplement was taken; on days 3 and 4, the midday samples were collected within an hour prior to supplementation (the classical, daily “trough” value for a drug). There was a significant daily pattern of variation in 25-hydroxyvitamin D concentrations (analysis of variance p ≤ 0.02 for 3 of the 4 days): peak midday mean 25-hydroxyvitamin D was approximately 20% higher than in the morning, and approximately 13% higher than in the evening. Trough sampling produced no significant difference in 25-hydroxyvitamin D compared to sampling an hour after the dose. An incidental finding was that acute illness during the study was related to acutely lower 25-hydroxyvitamin D at every sampling time in the day (p < 0.00001). Conclusions There was a consistent diurnal variation in 25-hydroxyvitamin D, with the peak at midday. There was no difference between trough versus post-dose blood draws. Acute illness may acutely lower serum 25-hydroxyvitamin D levels. Because within-person, within-day variability in 25-hydroxyvitamin D is approximately 20%, sampling time introduces systematic error in vitamin D nutritional assessment that is bigger than random analytical error or choice of assay method.