Longevity is associated with higher circulating levels of thyroid stimulating hormone (TSH) in the absence of differences in circulating thyroid hormones (TH), as previously observed in F2 members of long-lived families (F2-LLS) and their partners (F2-Con). The mechanism underlying this observed difference remains unknown.We hypothesized that the thyroid gland of members from long-lived families are less responsive to TSH stimulation, thereby requiring higher circulating TSH levels to maintain adequate TH levels.We performed a case-control intervention study with a single intra-muscular (gluteal) injection with 0.1mg recombinant human TSH (rhTSH) in a subgroup of 14 F2-LLS and 15 similarly aged F2-Con. They were followed-up for 4 days. No serious adverse events were reported. For analyses, we compared time trajectories of TSH and TH, and the ratio of TH to TSH using area under the curve (AUC) calculations.The AUC fT4/AUC TSH ratio was significantly lower in F2-LLS than in F2-Con (estimated mean (95%CI) 1.6 (1.2-1.9) and 2.2 (1.9-2.6), respectively, p=0.01). The AUC Tg/AUC TSH ratio was also lower in F2-LLS than in F2-Con (median (IQR) 2.1 (1.4-3.6) and 3.2 (2.7-7.4), respectively, p=0.04). We observed the same trend with the AUC fT3/AUC TSH ratio, although the difference was not statistically significant (estimated mean (95%CI) 0.6 (0.4-0.7) and 0.7 (0.6-0.8), respectively, p=0.07).The present findings show that members of long-living families have a lower thyroid responsivity to TSH compared to their partners.