Commonly measured metabolite and hormone concentrations used to describe the metabolic status of lactating cows undergo diurnal variation resulting in distinct patterns. Studies have shown that feeding events can modulate these diurnal patterns as cows respond to the nutrient intake. What is less clear is the extent to which cow genetics and diet interact to modify the diurnal patterns of specific nutritionally related metabolites and hormones. The objective of this study was to investigate diurnal patterns in circulating metabolite and hormone concentrations in divergent strains of Holstein-Friesian cows (North American, NA; and New Zealand, NZ) offered either fresh pasture (FP) or a total mixed ration (TMR). Plasma concentrations of growth hormone (GH), insulin-like growth factor-1 (IGF-1), glucose, insulin, β-hydroxybutyrate (BHBA), and non-esterified fatty acids (NEFA) were determined at 4-h intervals for a minimum of three consecutive days. All of the above metabolites and hormones exhibited within-day variability. Main effects of genetic strain and diet were observed for plasma IGF-1, and a strain by diet interaction was observed for GH. Time of day×diet interactions were observed for both glucose and insulin. Three-way interactions (time of day×diet×strain) were observed for BHBA and NEFA. These data indicate different levels of diurnal variation, with glucose, insulin, NEFA and BHBA having the largest daily variation. These diurnal patterns need to be considered in future investigations of these metabolites and hormones.