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Overtly impaired thyroid function affects skeletal and cardiac muscle function, reflected in increased circulating skeletal muscle enzyme levels (ie, creatine kinase [CK] and myoglobin [Mb]), prolonged ankle reflex time (ART), and altered systolic time intervals (STI). The response of these markers of muscular function to l-T4 replacement therapy in patients with subclinical hypothyroidism was evaluated in a prospective, double-blind study. Sixty-six women with subclinical hypothyroidism (thyroid-stimulating hormone [TSH] 12.9 ± 8.2 mU/L) were randomly assigned to receive l-thyroxine or placebo for 48 weeks. Sixty-three of the 66 women completed the study. Mb, CK, ART, and STI were measured at baseline and 48 weeks after l-thyroxine or placebo treatment, respectively. In patients with markedly elevated TSH levels at baseline (>12 mU/L), the ART decreased significantly after 48 weeks of l-thyroxine treatment (P = 0.01). There was no treatment effect on circulating CK or Mb levels. The STI were not altered in subclinical hypothyroidism and consequently remained unchanged by l-thyroxine treatment. ART is significantly reduced after l-T4 replacement therapy in patients with markedly elevated TSH levels at baseline. CK and Mb, representing surrogate markers of muscle function, as well as systolic cardiomuscular function, assessed by measurement of resting left ventricular systolic function, are not affected in subclinical hypothyroidism. This suggests a mildly affected, yet compensated neuromuscular dysfunction. Of various potential surrogate markers of muscular function, ART seems to reflect best the subtle changes of mild thyroid failure.