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Vitamin D deficiency has been implicated in numerous human diseases leading to an increased interest in assessing vitamin D status. Consequentially, the number of requests for vitamin D measurement keeps dramatically increasing year-on-year. Currently, the recognised best marker of vitamin D status is the concentration of the 25-hydroxyvitamin D (25(OH)D) in the blood circulation. While providing an accurate estimate of vitamin D status at the point in time of sampling, it cannot account for the high variability of 25(OH)Dconcentration. In this proof of concept study we set out to provide evidence that 25(OH)Dcan be extracted from hair samples in a similar fashion to steroid hormones. Two of the authors (L.Z. and M.H.) provided hair samples harvested from the crown area of the scalp and the third author (E.L.) provided beard samples. These samples, cut into 1 cm lengths, were weighed, washed and dried. 25(OH)D was extracted using a previously published steroid hormones extraction procedure. Blood samples were taken from the subjects at the same time all tissue samples were analysed using liquid-chromatography mass spectrometry. Hair samples showed presence of quantifiable 25(OH)Dwith concentrations ranging from 11.9–911 pg/mg. The beard sample had a concentration of 231 pg/mg. Serum levels of 25(OH)Dranged from 72–78 nmol/L. The results presented here confirm the feasibility of measuring 25(OH)Din hair samples. The findings warrant further validation and development and have the potential to yield valuable information relating to temporal trends in vitamin D physiology. View Full-Text