The aim of this study was to evaluate variations in plasma thyroid hormones and TSH during a standardized long term overfeeding protocol (4.2 megajoules/day [corrected] during a 100-day period) in 24 lean adults (12 pairs of monozygotic twins) and to assess their relationships with body composition and resting metabolic rate (RMR) changes. Compared to preoverfeeding values, basal plasma T3 concentrations were increased on day 25, but not later; basal plasma T4 and free T4 (FT4) concentrations were unchanged; basal plasma rT3 concentrations were persistently decreased throughout the entire protocol; and the TSH response to TRH stimulation was persistently enhanced. The TSH response to TRH before overfeeding was positively correlated with the changes in RMR with overfeeding (r = -0.53; P < 0.01). No association was found between changes in basal plasma T3 concentrations and changes in RMR. However, changes in basal T3 were positively related to changes in body weight (r = 0.46; P < 0.05). A significant within-pair similarity was found for changes in T4 and FT4 with overfeeding (P < 0.05). We conclude that 1) during overfeeding, the early increase in T3 concentrations is a transitory phenomenon, whereas the decrease in rT3 concentrations and the increased TSH response to TRH are more sustained; 2) the TSH responsiveness to TRH stimulation could be a predictor of the changes in RMR during times of increased energy intake; 3) there is no evidence for a direct role of T3 in the adaptation of resting energy expenditure during a long term overfeeding protocol; and 4) the genotype could be involved in the changes in T4 and FT4 during a prolonged positive energy balance period.