Fig. 2.

Overview of the study and intervention effects on the gut microbiome composition. a Graphical overview of the HELENA Trial design showing details of all data collected over the course of the trial. In total, 150 overweight/obese adults were randomly assigned to the ICR (5 days eucaloric diet, 2 days of ~25% energy requirement), CCR (daily reduction of ~20% of caloric intake) or CTR group, 147 of them provided stool samples at baseline. Anthropometric measurements were performed for all participants. Blood samples were collected at all study timepoints for the assessment of plasma/serum concentrations of routine biomarkers. Dietary information was collected at baseline, week 12, and week 50. The amount of weight loss was significantly higher among ICR and CCR participants compared to CTR participants at all study timepoints, as previously published by Schübel et al. [22]. Differences in weight change across intervention groups at each timepoint was assessed using linear mixed models adjusted for age and sex, with participant identifiers as random effects in the model. A significant difference, i.e., p < 0.05 is indicated by (*). ICR – intermittent calorie restriction; CCR – continuous calorie restriction; CTR – controls. b The gut microbiota was predominantly composed of bacteria belonging to Clostridia followed by Bacteroidia as shown by stacked bar charts of taxonomic classes across timepoints and subdivided by intervention group. c Non-metric multidimensional scaling (NMDS) plots of Bray-Curtis dissimilarity distances based on ASV relative abundances between samples after 12 weeks of the intervention. Each point represents the microbial community of a sample, and the colour indicates the intervention group to which it belongs. NMDS plots do not show clustering by intervention groups and PERMANOVA does not indicate a statistically significant difference between groups. d Intra-individual variability (same subject compared across time points) was significantly lower compared with inter-individual variability (different subjects, same group and different subjects, different groups). Technical replicates in this study showed a very low variation in Bray-Curtis dissimilarity distances based on ASV relative abundances, attesting to a high technical reproducibility. Statistical significance as indicated above boxplots was assessed by Wilcoxon signed-rank test. e There was no significant intervention effect on overall gut microbiota alpha diversity (Shannon Index) across all intervention timepoints. All boxplots show the interquartile ranges (IQRs) as boxes, with the median as a black horizontal line and the whiskers extending up to the most extreme points within 1.5-fold IQR. Additional file 3: Source data 2