This study aimed to characterise the physiological response of rabbits to feed withdrawal without stress caused by crating and transport to the slaughterhouse. A total of 72 recently weaned Grimaud rabbits were allocated into 12 cages, each with 6 rabbits (3 females and 3 males, to reflect commercial practices). A preslaughter feed withdrawal time (FWT) was randomly assigned to each of the 12 cages (t=0, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 h). Blood lactate and cortisol concentrations were measured at exsanguination. These observations did not indicate an elevated level of stress in the rabbits (P>0.05). The maximum Longissimus lumborum glycolytic potential was observed for rabbits that fasted for 3 and 6 h and was relatively stable from 127.78 to 139.04 μmol/g for rabbits with FWT longer than 12 h. As expected, gastrointestinal tract and stomach content weights were lower for rabbits with longer FWT (P<0.0001), while caecum weights did not (P=0.051). Rabbits with longer FWT had lower stomach pH and higher caecum pH (both P<0.0001). Metataxonomic 16S analysis revealed that FWT had a significant effect (all P<0.01) on microbiome beta diversity in faeces and caecum. The polymerase chain reaction analysis using specific primers revealed Enterobacteriaceae presence in the faeces of male rabbits only at 18 and 22 h. Our results suggest that the caecotrophic behaviour of rabbits allows them to be particularly resistant to hunger despite their small size. However, to limit Enterobacteriaceae shedding, the FWT should not exceed 18 h.