Seasonal variation in circulating 25-hydroxyvitamin D (25OHD) levels related to seasonal and inter-day fluctuation in sunlight ultraviolet irradiation, may lead to misjudgments concerning 25OHD status in individual workers around threshold levels. Here, to examine seasonal and inter-day variations in plasma 25OHD, we conducted a longitudinal study involving indoor daytime workers. Subjects were four male indoor daytime workers aged 32-57 yr working in Kawasaki City, Japan. Blood samples were obtained on six days within two two-week periods in February and October, 2008. Plasma 25OHD, serum intact parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D [1α,25(OH)₂D] were measured. Individual monthly mean 25OHD levels were 16-56% higher in October than in February (p=0.03), while individual monthly mean intact PTH levels were 15-41% lower in October (p=0.09). No seasonal change was observed in 1α,25(OH)₂D (p=0.62). Notably, nearly all measured 25OHD levels in February were lower than the reference value of 20 ng/ml. Our study identified the occurrence of seasonal variation in circulating 25OHD and intact PTH levels, even after accounting for inter-day variability, and hypovitaminosis D in wintertime in indoor daytime male workers in Japan. Due to this variability, single spot measurements of 25OHD may lead to misjudgment of workers’ vitamin D status.