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Background: Serum protein electrophoresis (SPE) reference intervals (RIs) have been evaluated in different horses, but no specific values are shown for equine breeds as previously described in other species (dogs, cats), and no studies have been performed on SPE in draft horses. Objectives: This study aimed to determine RIs for SPE in heavy draft horses (Italian Heavy Draft Horse‐IHDH) living in central Italy. A comparison between different physiologic states (pregnancy and no pregnancy) and ages (foals and adults) was executed. Materials and Methods: Blood samples were collected from 215 apparently healthy horses (mares, stallions, and foals). SPE (total proteins, albumin, α1‐, α2‐, β1‐, β2‐, and γ‐fractions, A/G) was evaluated in the Veterinary Teaching Hospital of the University of Perugia. RIs were determined using well‐described, modern analytical and statistical methods. The normality of distributions was assessed using the Anderson‐Darling test. Differences between subgroups were evaluated using the Mann‐Whitney U test. A P <.05 was considered statistically significant for all analyses. Results: Our results showed that IHDHs had increases in TPs and the α2‐, β1‐, β2‐, and γ‐fractions, and decreases in albumin, α1‐globulins, and A/G ratios compared with the data reported in other horses. We also found that foals had significantly higher α1‐globulins and significantly lower albumin concentrations, and A/G ratios compared with those of the adult horses. Conclusions: In the present study, SPE RIs using agarose gels have been determined for the first time in a large number of draft horses (represented by IHDH). The obtained results provide a basis for the further investigation of equine breeds with natural breeding, and the impact of age and physiologic states on SPE. [ABSTRACT FROM AUTHOR]