Abstract Background: Intermittent energy restriction (IER) is gaining popularity as a weight-loss strategy. However, the effect of short-term energy restriction on thyroid hormone dynamics is not well characterized. Methods: Nineteen healthy women age 23.36± 2.08 yr (mean ± SD) with normal baseline thyroid function and negative anti-thyroid antibodies underwent two 5-day interventions of a prescribed diet and identical standardized exercise in the early follicular phase of two menstrual cycles - neutral energy availability (NEA) 45 kCal/kg*LBM/d followed by deficient energy availability (DEA) 20 kCal/kg*LBM/d. Energy requirements were estimated as previously described (doi.org/10.1210/jendso/bvaa046.1468) and were used to generate a diet and exercise regimen for each participant. On day 5 of both interventions, body composition was assessed by BodPod®. Standardized NEA or DEA breakfast and lunch were provided as appropriate as well as a standardized NEA snack on both sampling visits. Blood sampling was performed for 8 hours starting at ~0800 h with measurement of TSH and growth hormone (GH) every 10 min, cortisol every 30 min, total T3 (TT3), reverse T3 (rT3) and total T4 (TT4) every 60 min, free T3 (FT3), free T4 (FT4) and TBG at the beginning and end of sampling. Liquid chromatography-tandem mass spectrometry (LC-MS) was used for measurements of all thyroid hormones, with the exception of TSH and TBG which were measured by ELISA as were GH and cortisol. Data were analyzed using ANOVA-RM and linear mixed models. Results are presented as mean or least squared mean ± sem. Results: Body mass index, bodyweight and % fat mass were not different between interventions. GH and cortisol were unaffected by DEA (p=0.46, p=0.63). TBG was not affected by time of day or dietary intervention (p=0.95, p=0.41). However, compared with NEA, TT3 (89.15 ± 2.89 vs 95.55 ± 2.89 ng/dL for DEA and NEA, respectively; p<0.0001) and TSH (0.92 ± 0.08 vs 1.03 ± 0.09 μIU/mL; p=0.0011) were lower after DEA, while TT4 (6.26 ± 0.25 vs 6.06 ± 0.25 μg/dL; p=0.04), FT4 (3.37 ± 0.26 vs 2.94 ± 0.25 ng/d;, p=0.0052) and rT3 (11.77 ± 0.58 vs 8.85 ± 0.51 ng/dL; p<0.0001) were higher. Regardless of dietary intervention, FT3 (p=0.0005), TT3 (p<0.0001), TT4 (p<0.0001) and TSH (p<0.0001) decreased across the day. Conclusion: Using LC-MS for as a more robust measure of thyroid hormones, we have now shown that changes in thyroid hormone dynamics occur after only 5 days of 55% energy restriction in the absence of alterations in body composition, cortisol, GH, TBG or the circadian pattern of thyroid hormone secretion. The decrease in TSH combined with the decrease in TT3 and increase in rT3 support the contribution of both central and peripheral mechanisms to these changes. Taken together these results provide support for a multi-level adaptation in thyroid hormone dynamics to conserve energy expenditure in response to short-term energy restriction.