We measured serum thyroxine (T4) and 3,3',5-triiodothyronine (T3) concentrations in hyperthyroid cats (hourly for 10 hours in 14 cats, and daily for 15 days in seven cats) to assess fluctuation in thyroid hormone levels. Over the 10-hour study period the coefficient of variation (CV) for serum T4 and T3 concentrations ranged from 6.4–22.6% (mean = 12.0 ± 4.8%) and from 9.6–33.1% (mean = 17.5 ± 6.3%), respectively. During the 15-day study period, CV for serum T4 ranged from 6.6–34.8% (mean = 18.4 ± 9.3%), while CV for serum T3 ranged from 7.8–31.0% (mean = 20.1 ± 8.6%). These CV values were significantly higher than the expected intra-assay CVs (T4 assay, 5.1%; T3 assay, 7.7%). In addition, some of the cats with mild hyperthyroidism showed one or more normal serum T4 and T3 values during the course of the respective study periods. There was no specific time during the 10-hour study period at which the cats consistently showed peak serum T4 or T3 concentrations. These results suggest that serum thyroid hormone concentrations are subject to a degree of fluctuation that exceeds the usual assay variation, and that cats with mild hyperthyroidism can, at a given time, exhibit normal serum T4 and T3 values. Therefore, a diagnosis of feline hyperthyroidism should not be excluded on the basis of the finding of a single normal serum T4 or T3 value in a cat with clinical signs and physical examination findings consistent with the disease.