Acute stress has been previously reported as a source of unexplained variation in in vivo studies, thus resulting in an increase in a number of animals used for experiments. The intraperitoneal (i.p.) injections, most commonly used in laboratory mice for general anaesthesia (GA) may act as acute stressors, both due to immobilization of the animals and, in particular due to the pain related to a needle insertion into the peritoneum. Therefore, this study evaluated the effects of two GA protocols with i.p. injection of anaesthetics, in which one protocol was preceded with inhalation anaesthetic allowing for animals’ insensitisation to acute stressors related to immobilization/i.p. injection. The effect of the two GA protocols was assessed based on the volume of urine present in the urinary bladder, as it was previously shown that urination is one of physiological responses to acute stress in rodents (Antoniadis and McDonald, 2001). This study was based on a “sharing scheme” and the samples were obtained from animals included in other experiments that were destined to be sampled under GA without recovery; only animals that fulfilled pre-set inclusion/exclusion criteria were selected. A total of 56 mice of three strains (C57BL/6, n=32, 39.5+2.5g; scid, n=18, 20.5+1.03g; CPU2VEGF, n=6, 36.2+4.1g) were included. In 24 mice (IP group) GA was carried out with a dissociative anaesthetic (ketamine) and α2-agonist (xylazine) in a 10:1 mixture administered with a single i.p. injection (1.2 ml/kg live body mass, LBM). For the remaining 32 animals (ISOIP group), anaesthesia was induced with volatile anaesthetic gas (5% isoflurane) in a drop-closed anesthetic chamber; when a loss of righting reflex was observed these animals were dosed with a single i.p. injection of a ketamine and xylazine mixture, as described above. Under GA, an abdominal incision was made through the linea alba for needle aspiration of total urine from the urinary bladder. Out of a total of 56, the bladder was empty in 17 of the animals. However, this included a significantly higher (p<0.0001, the Pearson chi-square test) number of mice from IP group (14) vs. ISOIP group (3). After exclusion of animals with empty bladder, the effect of anaesthesia protocol on collected urine volume normalized for LBM [μL/1 g LBW] was evaluated with two-way ANOVA with strain and anaesthesia protocol as independent variables. The effect of anaesthesia protocol on collected urine volume was significant (0.95 + 0.32 vs. 5.03 + 0.29 μL/1 g LBW, for IP and IOSIP group respectively, p < 0.001), while the effect of the strain was not significant (p = 0.861). The results of this study suggest that an induction with inhalable anaesthetics can reduce acute stress related to i.p. injections in laboratory mice thus offering a refinement to many experimental procedures carried out on those animals.