It has been largely accepted that dietary changes have an effect on gut microbial composition. In this pilot study we hypothesised that Ramadan fasting, which can be considered as a type of time-restricted feeding may lead to changes in gut microbial composition and diversity. A total of 9 adult subjects were included in the study. Stool samples were collected before (baseline) and at the end of the Ramadan fasting (after 29 days). Following the construction of an 16S rRNA amplicon library, the V4 region was sequenced using the Illumina Miseq platform. Microbial community analysis was performed using the QIIME program. A total of 27,521 operational taxonomic units (OTUs) with a 97% similarity were determined in all of the samples. Microbial richness was significantly increased after Ramadan according to observed OTU results (P=0.016). No significant difference was found in terms of Shannon index or phylogenetic diversity metrics of alpha diversity. Microbial community structure was significantly different between baseline and after Ramadan samples according to unweighted UniFrac analysis (P=0.025). LEfSe analysis revealed that Butyricicoccus, Bacteroides, Faecalibacterium, Roseburia, Allobaculum, Eubacterium, Dialister and Erysipelotrichi were significantly enriched genera after the end of Ramadan fasting. According to random forest analysis, the bacterial species most affected by the Ramadan fasting was Butyricicoccus pullicaecorum. Despite this is a pilot study with a limited sample size; our results clearly revealed that Ramadan fasting, which represents an intermittent fasting regime, leads to compositional changes in the gut microbiota.