Although heat shock proteins (Hsps) are primarily considered as being intracellular, this study identified the presence of Hsp72 in plasma from female Holstein-Friesian dairy cattle. Plasma samples were collected from the same animals at different ages and on different days after calving and accordingly divided into 5 age classes. The age classes were calves less than 235 days of age, young heifers between 235 and 305 days of age, older heifers between 305 and 560 days of age, cows early in lactation, and cows later in lactation. For a subsample of animals within each age class, replicate plasma samples were collected from 1 to 7 days apart to test whether the Hsp72 concentration levels are repeatable on this shorter timescale. Hsp72 was observed in plasma samples from animals of all 5 age classes. For animals with blood samples taken a few days apart, the repeatability (within age class) of the Hsp72 concentration was 0.52 ± 0.06. Age and days from calving significantly affected the Hsp72 concentration level. The highest Hsp72 level was observed in older heifers (305–560 days of age). The repeatability of Hsp72 concentrations across age classes within animal was 0.22 ± 0.06. High environmental sensitivity and negative genetic associations between production and health traits in this high-producing breed have been documented earlier. Hsp72 is believed to be strictly stress inducible, and the finding of Hsp72 in plasma indicates that even apparently healthy individuals may experience extrinsic or intrinsic stress (or both).