The same prebiotics have produced inconsistent effects on microbiota when evaluated in different batch fermentation studies. To understand the reasons behind these discrepancies, we compared impact of one prebiotic formula on the same inoculated fecal microbiota in two frequently used batch systems: phosphate-buffered saline (PBS, oligotrophic) and basal culture medium (BCM, eutrophic). The microbiota was monitored using 454 pyrosequencing. Negative controls (no prebiotic) of both systems showed significant shifts in the microbiota during fermentation, although their pH remained relatively stable, especially in BCM, with increases in Bilophila and Escherichia/Shigella but a decrease in Faecalibacterium. We identified prebiotic responders via redundancy analysis by including both baseline and negative controls. The key positive and negative responders in the two systems were very different, with only 8 consistently modulated OTUs (7 of the 28 positive responders and 1 of the 35 negative responders). Moreover, some OTUs within the same genus responded to the prebiotic in opposite ways. Therefore, to obtain a complete in vitro evaluation of the modulatory effects of a prebiotic on microbiota, it is necessary to use both oligotrophic and eutrophic systems, compare treatment groups with both baseline and negative controls, and analyze the microbiota changes down to the OTU level.