Direct evidence in humans for the impact of the microbiome on nutrient absorption is lacking. We conducted an extended inpatient study using two interventions that we hypothesized would alter the gut microbiome and nutrient absorption. In each, stool calorie loss, a direct proxy of nutrient absorption, was measured. The first phase was a randomized cross-over dietary intervention in which all participants underwent in random order 3 d of over- and underfeeding. The second was a randomized, double-blind, placebo-controlled pharmacologic intervention using oral vancomycin or matching placebo (NCT02037295). Twenty-seven volunteers (17 men and 10 women, age 35.1 ± 7.3, BMI 32.3 ± 8.0), who were healthy other than having impaired glucose tolerance and obesity, were enrolled and 25 completed the entire trial. The primary endpoints were the effects of dietary and pharmacological intervention on stool calorie loss. We hypothesized that stool calories expressed as percentage of caloric intake would increase with underfeeding compared with overfeeding and increase during oral vancomycin treatment. Both primary endpoints were met. Greater stool calorie loss was observed during underfeeding relative to overfeeding and during vancomycin treatment compared with placebo. Key secondary endpoints were to evaluate the changes in gut microbial community structure as evidenced by amplicon sequencing and metagenomics. We observed only a modest perturbation of gut microbial community structure with under- versus overfeeding but a more widespread change in community structure with reduced diversity with oral vancomycin. Increase in Akkermansia muciniphila was common to both interventions that resulted in greater stool calorie loss. These results indicate that nutrient absorption is sensitive to environmental perturbations and support the translational relevance of preclinical models demonstrating a possible causal role for the gut microbiome in dietary energy harvest.