Vitamin A deficiency is common in pond sliders, and vitamin E and B1 deficiencies have also been described in carnivorous aquatic species such as crocodiles, but otherwise little is known about vitamin concentrations in the blood of reptiles, and reference intervals only exist for a few vitamins in a few species. The aim of this study was therefore to measure vitamins A, B1, B2, B6, B9, B12, and E in the heparinized whole blood and plasma of pond sliders (Trachemys scripta) (n = 188) from April to September 2022 using high-performance liquid chromatography (HPLC) and spectrophotometry. In addition to establishing reference intervals, possible influencing factors, including the season of blood sampling and the sex of the turtles, were evaluated. The median concentrations measured were as follows: vitamin A—0.17 mg/L, B1—59.10 µg/L, B2—693.50 µg/L, B6—2.375 µg/L, B9—16.06 ng/mL, B12—3225.00 pg/mL, and E—7.59 mg/L. Significant (p ≤ 0.05) seasonal variations were found for vitamin A, B1, and B9, and sex-specific variations were found for vitamin E. The results show that factors such as sex and the time of year play a role in vitamin levels in the blood of turtles and should be considered when interpreting blood results. The reference intervals established in the present study serve as a basis for further studies and help to control and improve the vitamin supply of the turtles, contributing to the better husbandry and nutrition of this species in human care.

Some trace elements are essential for the health of vertebrates, but little is known about their function, the amounts required, and the factors influencing their metabolism in tortoises. The aim of this study was therefore to measure trace elements (chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), molybdenum (Mo), selenium (Se), zinc (Zn)) in heparinized blood plasma of Hermann’s tortoises (Testudo hermanni) (n = 520) from March to September 2022 using inductively coupled plasma mass spectrometry (ICP-MS) and to establish specific reference intervals. Additionally, the influence of sex, season, and region of sample collection on the measured values were evaluated. Significant (p ≤ 0.05) sex-specific differences were found for Cu, Mg, and Mn; seasonal differences were found for Cr, Cu, Mn, Mo, and Se; and the region in which the tortoises were kept significantly impacted Cr, Cu, Fe, Mg, Mo, and Se levels. The results show that all of these factors should be consider when establishing and interpreting blood trace element levels in tortoises.