In this study, we investigated the effects of short-term energy loads on changes in gut microbiome-derived tryptophanase and fecal serotonin levels and their association with variations in energy absorption. This randomized crossover energy-load intervention study included 15 healthy participants subjected to three dietary conditions - overfeeding, control, and underfeeding - for eight days. The effects of the dietary conditions on energy absorption (digestible and metabolizable energy) were assessed using a bomb calorimeter. Fecal serotonin levels were assessed using LC-MS/MS, and the gut microbiota was analyzed using the 16S rRNA gene and metagenomic shotgun analysis. Significant differences were observed in digestible energy (p < 0.001), with higher values in the overfeeding than in the control (p = 0.032) conditions. Furthermore, significant differences were noted in metabolizable energy and gut transit time (p < 0.001), both of which were higher in the overfeeding than in the control (metabolizable energy: p = 0.001; gut transit time: p = 0.014) and underfeeding (metabolizable energy: p < 0.001; gut transit time: p = 0.004) conditions. Fecal serotonin levels differed significantly (p < 0.001), with significantly lower levels in the overfeeding than in the control (p = 0.005) and underfeeding (p < 0.001) conditions. Tryptophanase exhibited significant differences (p = 0.0019), with lower gene abundance in the overfeeding than in the underfeeding (p = 0.001) condition. Tryptophanase positively correlated with Bacteroides abundance under all conditions (correlation coefficient: 0.696-0.896). Intra-individual variability in fecal serotonin levels was significantly negatively associated with digestible energy (β = -0.077, p = 0.019). The findings suggest that short-term energy loads dynamically alter fecal serotonin, Bacteroides, and tryptophanase levels. Moreover, changes in fecal serotonin levels might be indirectly associated with energy absorption.