This study shows, for the first time, the absence of a vital effect in the clumped isotope carbonate (Δ₄₇) fossil ostracod signal and confirms the ability of the novel ostracod-Δ₄₇ thermometer to reconstruct past temperatures and hydrological conditions in complex lacustrine systems. Furthermore, the application of Δ₄₇ analyses on the ostracod species Candona angulata and Cyprideis torosa from Lake Trasimeno record (central Italy), which today precipitate their shells during the cold and the warm season respectively provides evidence that by combining biological (i.e., ostracod shell precipitation timing), paleontological (i.e., identification of ostracod species) and geochemical (i.e., Δ₄₇) approaches, the ostracod-Δ₄₇ thermometer accurately reconstructs past seasonality. Despite the absence of a vital effect, not all species can be combined for Δ₄₇ analyses in environments with seasonal temperature variations; only those that precipitate their shells during the same season should be considered. The application of the ostracod-Δ₄₇ thermometer on the Trasimeno lacustrine record gives rise to the first continental warm season paleotemperature reconstruction of the last 43 ky in central Mediterranean area. The combination of Δ₄₇ and oxygen isotope composition (δ¹⁸O_ost) measured on ostracod shells provides the isotopic composition of the water from which the carbonate precipitated (δ¹⁸O_w) and thereby, changes in the evaporation/precipitation balance in this area. Before the Last Glacial Maximum (LGM), equivalent to the Marine Isotopic Stage 3 (MIS3, from 43 to 29ky), warm season temperatures ranged from 15 ± 1.6 °C to 22 ± 2.3 °C, equivalent to 2 to 6 °C colder than today. Hydrological conditions during this period are similar to the present-day ones, characterized by a permanent lake and a high evaporation/precipitation ratio (E/P). The drastic decrease of the warm season temperatures (ranging from 10 ± 2.9 °C to 17 ± 3.1 °C) and of the E/P ratio during LGM and Late-glacial (MIS2, from 29 to 11.6 ky) correspond to the global climate cooling and low summer insolation, suggesting an amplifying role, of the latter, in the effects of the millennial scale climatic variations. At the Pleistocene/Holocene transition, both warm season temperature (25 ± 2 °C) and the E/P ratio increased in conjunction with the summer insolation. During the early Holocene, warm season temperature (23 ± 2 °C) closely resembles present-day values. However, cold season temperature (12 ± 2 °C) is approximately 4 °CC warmer than today. Notably, no hydrological differences are identified between the warm and the cold season underlying a lower seasonality contrast compared to the present, along with enhanced warm season precipitation. The good agreement between the Δ₄₇ temperatures reconstructed for the last 1 ky and the temperatures presently recorded at Lake Trasimeno (8 °C cold and 22 °C for warm season), confirms the accuracy of the analyses and the applicability of the ostracod-Δ₄₇ thermometer to reconstruct seasonal temperature changes.