Here, we tested the changes occurring in several plasma analytes during different stages of the lactation cycle of high welfare raised multiparous Holstein cows, and provided reference intervals (RI) for plasma analytes concentrations. Eleven high-welfare farms (HWF) located in Northern Italy were selected and their herds used to recruit 361 clinically healthy cows undergoing the dry (from −30 to −10 days from real calving; DFC), the postpartum (from 3 to 7 DFC), the early lactation (from 28 to 45 DFC) and the late lactation phases (from 160 to 305 DFC). Cows affected by subclinical diseases (SCD) were retrospectively excluded, and a subset of 285 cows was selected. Data of plasma analytes underwent ANOVA testing using physiological phases as predictors. The individual effect of each phase was assessed using a pairwise t-test assuming p ≤ 0.05 as a significance limit. A bootstrap approach was used to define the reference interval (RI) for each blood analyte within physiological phases having a pairwise t-test p ≤ 0.05. The concentration of nonesterified fatty acids, albumin, cholesterol, retinol, paraoxonase and tocopherol changed throughout all the physiological phases, whereas the concentration of K, alkaline phosphatase and thiol groups remained stable. Triglycerides, Zn, and ferric ion reducing antioxidant power in the dry phase and BHB, Ca, myeloperoxidase, haptoglobin, reactive oxygen metabolites and advanced oxidation of protein product in postpartum differed compared with other physiological phases. During the dry phase, Packed cell volume, Cl, and urea concentrations were similar to during the postpartum phase. Similarly, Na, γ-glutamyl transferase and β-carotene concentrations were similar to during the early lactation phase; fructosamine and bilirubin concentrations were similar to during the late lactation phase. During the postpartum phase, fructosamine and P concentrations were similar to during the early lactation phase, and the aspartate transaminase concentration was similar to during the late lactation phase. During the early lactation phase, Mg, creatinine, total protein, globulin and ceruloplasmin concentrations were similar to during the postpartum phase, while the urea concentration was similar to during the late lactation phase. All these plasma analytes differed among the other phases. This study identifies physiological trends affecting plasma analytes concentrations during the different stages of the lactation cycle and provides a guideline for the duration and magnitude of their changes when animals are healthy and raised in optimal welfare conditions.