The metabolism of tortoises is influenced by many factors, which in turn also influences the clinical chemistry parameters in these animals. There are currently limited data available on species, sex, and season-specific reference intervals. The goal of this study was to establish new reference intervals for adult Hermann's tortoises (Testudo hermanni), which include variations according to season and sex. Lithium heparinized blood samples from 256 Hermann's tortoises were collected and biochemistry parameters (alkaline phosphatase [ALP], glutamate-dehydrogenase [GLDH], alanine–aminotransferase [ALT], aspartate–aminotransferase [AST], bile acids [BA], creatine kinase [CK], total protein [TP], albumin [Alb], urea, uric acid [UA], inorganic phosphorus [P], total calcium [Ca], sodium [Na], and potassium [K]) were measured from May 2016 to October 2017. The results showed many variations depending on sex and season. Male Hermann's tortoises had higher ALT, BA, CK, and UA concentrations, and females had higher Ca concentrations. The concentrations of BA, UA, GLDH, TP, and Alb decreased during the course of the year, and ALT increased over time in males. The present study demonstrates that it is important to establish specific reference intervals for each species that include seasonal and sex specific variations in order to facilitate correct interpretation of blood results.