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Serum metabolic profile is a common method to monitor health and nutritional status of dairy cows, but blood sampling and analysis are invasive, time-consuming, and expensive. Milk mid-infrared spectra have recently been used to develop prediction models for blood metabolites. The current study aimed to investigate factors affecting blood β-hydroxybutyrate (BHB), non-esterified fatty acids (NEFA), and urea nitrogen (BUN) predicted from a large milk mid-infrared spectra database. Data consisted of the first test-day record of early-lactation cows in multi-breed herds. Holstein-Friesian cows had the greatest concentration of blood BHB and NEFA, followed by Simmental and Brown Swiss. The greatest and the lowest concentrations of BUN were detected for Brown Swiss and Holstein-Friesian, respectively. The greatest BHB concentration was observed in the first two weeks of lactation for Brown Swiss and Holstein-Friesian. Across the first month of lactation, NEFA decreased and BUN increased for all considered breeds. The greatest concentrations of blood BHB and NEFA were recorded in spring and early summer, whereas BUN peaked in December. Environmental effects identified in the present study can be included as adjusting factors in within-breed estimation of genetic parameters for major blood metabolites.