The European wildcat (Felis silvestris), particularly its Iberian lineage, is experiencing an important population decline due to various threats. For this reason, we started in 2017 an intensive monitoring of a population of this species in southeastern Spain. To determine their physiological status, we conducted health checks on wildcats during 2017-2023. We captured and anesthetized 10 males (six adults and four non-adults) and 14 females (eight adults and six non-adults) for the collection of biometric measurements and biological samples through a standardized handling protocol. All sampled wildcats were monitored after their release through radio-tracking and/or camera-trapping for at least three months. The collected samples were used to perform morphological, hematological, biochemical, and endocrine analyses. We found that all the physiological parameters fell within previously established ranges (when available) and may serve as a reference for future studies on the wildcat. The studied Iberian wildcats were larger than the wildcats from temperate Europe, which do not conform to Bergmann's rule. This could be the result of an adaptation of Iberian wildcats to capture larger prey (i.e., European rabbit Oryctolagus cuniculus) in the Mediterranean region, and calls for a reassessment of the taxonomic status of the Iberian lineage of this felid. Finally, as revealed by the monitored individuals, the handling protocol proved to be optimal, resulting in a practical guidance for further field surveys.