© 2019 The Authors. Experimental Physiology © 2019 The Physiological Society New Findings: What is the central question of this study? Does the link between cardiorespiratory fitness and gut microbiota diversity persist after adjusting for the potential effects of percentage body fat and activity-related energy expenditure (AEE)? What is the main finding and its importance? This is the first study to examine the link between cardiorespiratory fitness and gut microbiota diversity while accounting for the underlying effects of percentage body fat and free-living AEE. Results from the present work suggest that cardiorespiratory fitness, not physical activity, is a superior correlate of gut microbiota diversity among post-primary treatment, non-metastatic breast cancer survivors. Abstract: Cancer treatment uniquely triggers multiple physiological shifts detrimental to overall health. Although previous research indicates a link between the gut microbiota and cardiorespiratory fitness, it is unclear whether these findings are attributable to potential underlying effects of percentage body fat or free-living activity energy expenditure (AEE). The microbe composition of faecal specimens from 37 breast cancer survivors was determined using 16S microbiome analyses. Individual-sample microbiota diversity (α-diversity) and between-sample community differences (β-diversity) were examined. Peak oxygen uptake ((Formula presented.)) was estimated from a graded exercise test consistent with the modified Naughton protocol, in which exercise terminates at 85% of age-predicted maximal heart rate. The AEE was measured over 10 days using doubly labelled water, wherein the percentage body fat was calculated from total body water. Pearson correlations revealed α-diversity indices (Chao1, observed species, PD whole tree and Shannon) to be positively associated with (Formula presented.) (r = 0.34–0.51; P < 0.05), whereas the percentage of maximal heart rate during stages 1–4 of the graded exercise test (r = −0.34 to −0.50; P < 0.05) and percentage body fat (r = −0.32 to −0.41; P < 0.05) were negatively associated with the same α-diversity indices. Multiple linear regression models showed that (Formula presented.) accounted for 22 and 26% of the variance in taxonomic richness (observed species) and phylogenic diversity after adjustment for percentage body fat and menopausal status. Unweighted UniFrac (β-diversity) was significant for several outcomes involving cardiorespiratory fitness, and significant taxa comparisons were found. Associations between gut microbiota and free-living AEE were not found. Results from the present work suggest that cardiorespiratory fitness, not physical activity, is a superior correlate of gut microbiota diversity.