In non-ruminant species, glucose tolerance and insulin sensitivity are known to be regulated by circadian rhythms, which are repeating ~24 h cycles that govern many aspects of behavior, physiology, and metabolism. However, it is unknown if these rhythms exist in dairy cows. Our objective was to determine the fit of a daily rhythm of glucose, insulin, and nonesterified fatty acid clearance rates independent of daily patterns of nutrient intake. To accomplish our objective, 12 multiparous lactating Holstein cows were enrolled in a within-subject design conducted over two experimental periods (n = 6/period). Within each period, cows were subjected to intravenous glucose tolerance tests (IVGTT) representing 0300, 0900, 1500 and 2100 h. The 0900 and 2100 h IVGTT were performed 36 h apart, followed by a 7-d washout and 1500 and 0300 h IVGTT performed 36 h apart. Cows were fed 12x/d at 2 h intervals beginning 24 h prior to the first IVGTT in each set until the second IVGTT in each set to stabilize feed intake across the day, with 1x/d feeding occurring during the washout period. For each IVGTT, 250 g of glucose were infused as a 50% w/v D-glucose solution via a jugular catheter and blood was collected at -15, -5, immediately before, 0, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min relative to infusion. A linear model with the fixed effects of cosine and sine and random effect of cow within period was used for the outcomes of clearance rate, half-life, baseline concentration, time to baseline concentration, and area under the curve (AUC) for glucose, insulin, and NEFA. A zero-amplitude test was used to determine the fit of a 24 h cosine function and cosinor rhythmometry was used to determine the amplitude and acrophase of the 24 h rhythm. Insulin concentrations at baseline followed a diurnal rhythm. Glucose and insulin clearance rate, half-life, and AUC also followed a diurnal rhythm. Glucose and insulin clearance rates peaked at 1247 h and 0944 h, respectively. No circadian rhythm was detected for plasma NEFA concentrations. Results suggest that insulin-stimulated glucose uptake is controlled differently throughout the day by circadian rhythms.