Evaluating bovine udder physiological status requires reliable diagnostic markers that remain consistent across varying farm management practices. This study aimed to characterize milk fatty acid (FA) patterns and total polymorphonuclear neutrophils (PMN) to determine their potential as combined markers for udder health and physiological status. A total of 31,463 milk test records from 18 commercial dairy herds were analyzed using Fourier transform mid-infrared spectroscopy and automated flow cytometry to evaluate milk FA composition, somatic cell counts, and differential somatic cell counts. Mixed linear models assessed the effects of season, parity, days in milk, and milk yield on FA and PMN variability. Measuring de novo FA as a relative proportion reduced herd-level variance from 23.5% to 1.8% compared to concentration measurements. De novo FA proportions peaked at 91–120 days in milk and reached their lowest levels during summer. Among healthy cows (somatic cell count ≤ 50,000 cells/mL), a lower de novo FA proportion (≤ 22%) significantly correlated with elevated total PMN counts in spring and summer. Integrating relative de novo FA proportions with PMN counts may provide a robust, farm-independent diagnostic tool to assess udder physiological status and immune capability for improved herd management.