This study was designed to address the question of whether decreased hypothalamic TRH secretion was responsible for the transient decline in serum TSH levels characteristic of the early phase of fasting in man. Changes in serum TSH, total T4 (TT4), free T4 (FT4), and total T3 (TT3)/TT4 and total rT3(TrT3)/TT4 ratio values together with plasma TRH levels were evaluated before, during, and after a constant 48-h infusion of either TRH (75 ng/min), or saline, initiated at 10 h into a 6-day fast, in groups of six ambulating, mildly obese female subjects. On the first day of the fast, no change in serum TSH levels was seen with saline infusion relative to control, whereas mean TSH levels rose 2 1/2-fold with TRH infusion. On the second day of the fast, serum TSH levels simultaneously declined in both the saline- (43%, P less than 0.005) and TRH- (52%, P less than 0.005)-infused groups relative to the previous day. On the third day of the fast, after stopping the infusions, mean serum TSH levels fell below control in both the saline- and TRH-treated groups, but subsequently returned to prefasting levels. A distinct circadian pattern of TSH release was present throughout the study. Plasma TRH values were unaffected by fasting in the saline-infused group, but rose 2 1/2-fold in the TRH-infused group. The sequence of changes in the serum thyroid hormone indices were similar for the two groups: a rapid rise in FT4 was followed by a gradual fall in TT3/TT4 ratios during the first 24 h of fast, which was followed by a rise in TrT3/TT4 ratios after 48 h of fast. This study in fasting, mildly obese females suggests that: 1) The transient suppression of serum TSH during early fasting is not TRH mediated. 2) Fasting does not alter plasma TRH levels. 3) A temporal sequence of changes in serum thyroid hormone indices occurs in fasting, this being an initial rise in FT4 (10 h) followed by a fall in both serum TT3/TT4 (12-14 h) and TSH (30-36 h) and finally by a rise in TrT3/TT4 levels (48 h). This sequence of events suggests that the initial inhibition of serum TSH levels in early fasting results from the acute elevation in FT4 levels, and that the reestablishment of normal serum TSH levels with continued fasting is associated with declining serum TT3 levels.