Background: Although several myocardial iodine 123 metaiodobenzylguanidine (MIBG) indices are increasingly used to detect alterations in myocardial sympathetic activity in various forms of cardiac pathology, published measurements of normal values and within-subject variability are lacking. Methods and Results: Twenty-five healthy volunteers underwent planar and single photon emission computed tomography (SPECT) imaging. Heart-mediastinum ratio (H/M) and myocardial washout were calculated from planar images comparing three different methods for the assessment of myocardial activity: (1) global region over the myocardium (cavity included), (2) global region over the myocardium (cavity excluded), and (3) fixed small myocardial region. Segmental (relative) uptake and washout were assessed by SPECT. For all MIBG indices, the interindividual variation was the lowest for methods 1 and 2. In SPECT this variation was low for relative segmental uptake compared with washout. In 9 subjects a second MIBG scintigraphy was performed after 3 months. The within-subject variability of H/M and washout assessed by planar methods 1 and 2 was 5%, whereas it was approximately 9% for planar method 3. For relative segmental uptake from SPECT, this variability was 5%. Conclusion: MIBG H/M (planar) and relative segmental uptake (SPECT) show a low interindividual and within-subject variability. This enables the detection of small (regional) variations in myocardial sympathetic nervous function, especially to monitor the effect of therapeutic interventions in patients with various cardiac diseases. (J Nucl Cardiol 2004;11: 126-33.