Ectothermic organisms often experience considerable variation in their body temperature throughout the circadian cycle. However, studies focusing on the measurement of physiological traits are usually performed under constant temperature regimes. This mismatch between thermal exposure in the field and experimental conditions could act as a stressor agent, since physiological functions are strongly influenced by temperature. Herein, we asked the question whether constant thermal regimes would cause a stress response and impact the immunity of the South American rattlesnake (Crotalus durissus) when compared to a fluctuating thermal regime. We addressed this question by determining heterophil:lymphocyte ratio (H:L), plasma bacteria killing ability (BKA) and corticosterone levels (CORT) in snakes kept under a constant temperature regime at 30°C, and under a fluctuating regime that oscillated between 25°C at nighttime to 35°C at daytime. The experiments had a mirrored design, in which half of the snakes were subjected to a fluctuating-to-constant treatment, while the other half was exposed to a constant-to-fluctuating treatment. The shift from constant to fluctuating thermal regime was accompanied by an increase in plasma CORT levels indicating the activation of a stress response. Exposure to a fluctuating thermal regime at the onset of the experiments induced a decrease in the BKA of rattlesnakes. H:L was not affected by treatments and, therefore, the shift between thermal regimes seems to have acted as a low intensity stressor. Our results suggest that the removal from temperatures close to the snakés preferred body temperature triggers a stress response in rattlesnakes.