Background While limited data suggest that the fecal microbiota in healthy people are stable over time, the intra-individual variability of the fecal microbiota in constipated patients is unknown. Methods This study evaluated the intra-individual reproducibility of fecal microbiota analysed with 16S rRNA gene sequencing in two stool samples collected without and after a laxative, respectively, in 25 healthy people and 25 constipated women. Participants completed a food record for 3 days before the stool collection. Colonic transit was measured with scintigraphy. Key Results The constipated patients were older (48±15 versus 39±10 years, P=.02) than healthy participants but had a similar BMI. The total daily caloric intake was less (P=.005) in constipated (1265±350 kcal) than healthy participants (1597±402 kcal). Fourteen patients but only 2 controls (P<.005), had delayed colonic transit. For most measures of alpha (e.g., Observed OTU number, Shannon index) and beta diversity (e.g., Bray-Curtis dissimilarity, UniFrac, phyla level abundance), the ICCs between 2 stool samples were high, indicating moderate or strong agreement, and similar in healthy people and constipated patients. The ICC for the weighted UniFrac distance, which is weighted by abundance, was lower than its unweighted counterpart, indicating that the unweighted measure is more robust and reproducible. Conclusions & Inferences The intra-individual reproducibility of fecal microbiota in constipated patients is high and comparable to healthy participants. For most purposes, evaluating the fecal microbiota in a single stool sample should generally suffice in adequately powered studies of healthy and constipated patients.