Determining whether associations between gut microbiota characteristics and host physiology represent causal relationships is a fundamental challenge for microbiome research. We report a detailed investigation of microbiome assembly in C57BL/6 germ-free mice across a period of 70 days and compare the effects of single and multiple rounds of gavage, using both native and antibiotic-disrupted murine donor material. Recipients of the native microbiota did not achieve compositional stability until day 28 and persistent differences to donor microbiota remained until day 70. Performing multiple rounds of gavage significantly increased the cumulative number of detected taxa (mean increase: 10.4%) and compositional similarity to donor, and significantly reduced within-group variance (p < 0.05). Multiple rounds of gavage with antibiotic-disrupted microbiota provided no substantial benefit in relation to compositional similarity to donor or within-group variance. The process of donor microbiota establishment in recipient animals is necessary before experimentation commences and is considerably influenced by donor microbiota characteristics. Display omitted •Gut microbiota in germ-free mice stabilize 28 days after native microbiota gavage•Repeated gavage of native microbiota increases similarity to donor microbiota•Establishment of antibiotic-disrupted microbiota does not improve with repeated gavage•Donor microbiota characteristics influence fidelity of microbiota re-establishment Biological Sciences; Microbiology; Microbiome