Thyroid hormones and the factors influencing them are rarely studied in tortoises. This study therefore aimed to calculate reference intervals (RI) for specific species, sexes, and seasons for thyroid hormones and iodine levels in blood of four adult Mediterranean tortoise species and to evaluate possible correlations between thyroid hormones, serum iodine, plasma protein and increased liver and kidney values. Thyroid hormones (total tetraiodothyronine [tT4], free tetraiodothyronine [fT4], total triiodothyronine [tT3], and free triiodothyronine [fT3]) were measured in plasma from adult, healthy Hermann´s (Testudo hermanni, n = 255), spur-thighed (Testudo graeca, n = 89), marginated (Testudo marginata, n = 72), and Russian tortoises (Testudo horsfieldii, n = 30). Species, sex and season specific variations were determined by Bayesian information criterion (BIC) and correlations between plasma thyroid hormones, protein, iodine and increased liver/kidney values were evaluated by Spearman’s rank correlation test. Total T4 did not reveal any species, sex, or seasonal differences (RI 0.102 to 0.455 µg/dL), while seasonal differences were found for fT4 (RI spring 0.624 to 9.012; summer 0.379 to 5.476; fall 0.376 to 5.426 pmol/L). The tT3 levels differed significantly depending on species, season, and the interaction of species x season. Seasonal differences were also found for fT3 and iodine. Several significant (p < 0.05) correlations were detected between the tested analytes, especially positive correlations between tT4 and fT4. These results provide a tool for the evaluation of thyroid hormone levels in Mediterranean tortoises and indicate the influence of season on the thyroid in these animals.

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