Subclinical hypothyroidism (SCH) is a common medical condition affecting 4-10% of the population. Unlike overt hypothyroidism, clinical manifestations are unclear and treatment remains controversial. It is known that fatigue may improve with levothyroxine in these patients but the mechanisms linking symptoms with abnormal tissue function are poorly understood. It was hypothesized that fatigue in patients with SCH is caused by peripheral tissue functional changes and that these changes are reversible with levothyroxine treatment. The objective of the study was to quantify the specific abnormalities in cerebral blood flow, cardiac function, cardiac and muscular energetic function, and autonomic function in patients with SCH, and to measure the changes in these abnormalities after levothyroxine therapy with any associated impact on fatigue. This was a pilot study as no previous studies looking into the mechanism of fatigue in patients with SCH have been reported. Subjects with SCH (TSH 4.0 -10.0 mU/L, normal free T4) and fatigue were studied before and after levothyroxine therapy and were compared with age and gendermatched healthy controls (HC). Cerebral blood flow (CBF) was measured by MR arterial spin labelling. Cardiac function was measured using impedance cardiography. Cardiac and calf muscle energetic functions were measured by 31-Phosphorous Magnetic Resonance Spectroscopy. Autonomic function was assessed using heart rate variability. At baseline, patients with SCH had increased CBF, impaired cardiac function, and lower cardiac and calf muscle energetic function, compared with HC. Autonomic function was equal to that of HC. After levothyroxine treatment, CBF decreased, cardiac function was unchanged, and cardiac energetic function improved. Calf muscle energetic function did not improve but autonomic function tests did. Although fatigue improved after levothyroxine treatment, these improvements were not correlated with peripheral tissue functional changes. Novel physiological abnormalities in both CBF and cardiac and calf muscle energetic functions have been demonstrated by these studies. Improvements were seen in CBF, ii cardiac energetic function and autonomic function after levothyroxine treatment. These parameters may play a role in the reduction of fatigue and warrant further investigation.