Eleven patients with treated hypothyroidism were investigated to examine the effects of time on their thyroid function tests. Each patient was clinically and biochemically euthyroid on once daily thyroxine replacement therapy, taken in the morning. TSH followed a diurnal rhythm with a peak level at 23.30 h and a trough level at 14.30 h. Four subjects had TSH trough levels within the normal range, but with peak levels outside this range. FT4 and FT3 levels fell from their highest levels some three hours after ingestion to the lowest levels just prior to the next dose. This study shows that there are significant time-related variabilities in TSH and thyroid hormone levels in treated hypothyroid patients. This should be taken into account when interpreting results of their thyroid function tests.