<p>Anesthesia is indispensable for minimizing stress during invasive procedures. However, anesthesia can induce undesired effects on the organism and its physiological homeostasis. In experiments involving animals, these consequences may reduce animal welfare and alter experimental outcomes. Moreover, most of the studies characterizing the effects of murine anesthetic protocols on animal welfare do not explore the impact of prolonged anesthesia for procedures lasting more than 2 h. Here we investigated the effect of prolonged anesthesia on vital parameters and immune responses to vaccination, comparing isoflurane (Iso), ketamine–xylazine (KX), and KX with oxygen supplementation (KXO₂), in the presence or absence of buprenorphine (BPP). KX induced hypoxia and 100% mortality, which were prevented by oxygen supplementation (KXO₂) and were not associated with BPP. By contrast, Iso induced safe and fast induction and recovery. Furthermore, we investigated the effects of these protocols on the immune responses and motility of immune cells following vaccination. The results showed that KX reduced immune cell numbers and increased cell death, complemented by elevated levels of the inflammatory proteins IL-6 and IFNγ. In addition, KX altered the motility patterns of T cells, B cells and neutrophils, potentially influenced by hypoxia. Conversely, Iso exhibited fewer immune artifacts, regardless of BPP. These findings highlight the importance of evaluating anesthesia protocols with respect to animal welfare and experimental reproducibility, especially for immunological studies. Oxygen supplementation emerged as an important refinement to mitigate hypoxia in KX. Iso showed superior safety and fewer artifacts compared with KX and KXO₂, demonstrating its suitability for immunological research and intravital microscopy.</p>