Relationships between human gut microbiota, dietary habits, and health/diseases are the subject of epidemiological and clinical studies. However, the temporal stability and variability of the bacterial community in fecal samples remain unclear. In this study, middle-aged Japanese male and female volunteers (n = 5 each) without disease were recruited from the Sakura Diet Study. Fecal samples and lifestyle information were collected in every quarter and at each defecation for 7 continuous days. Next-generation sequencing of 16S rDNA and hierarchical clustering showed no time trend and intra-individual differences in both fecal sample sets. Significant inter-individual variations in seasonal and daily fecal sample sets were detected for 24 and 23 out of 39 selected dominant genera (>0.1% of the total human gut microbiota; occupation rate >85%), respectively. Intra- to inter-individual variance ratios in 26 and 35 genera were significantly <1.0 for seasonal and daily stabilities. Seasonal variation in fermented milk consumption might be associated with Bifidobacterium composition, but not with Lactobacillus. For most of the dominant genera in the human gut microbiota, inter-individual variations were significantly larger than intra-individual variations. Further studies are warranted to determine the contributions of human gut microbiota to nutritional metabolism, health promotion, and prevention/development of diseases.