In preclinical safety studies in which the administration of a test compound causes reductions in food consumption, body weights, and organ weights, it may be difficult to differentiate direct compound-induced effects on organ weights from those simply due to reduced nutrition. To address this problem in reference to the heart, hearts were obtained from rats that were known to have had reductions in body weights and absolute heart weights as a result of feed restriction. Rats (40/sex) were divided into 4 groups (10/sex) and given quantities of ad libitum diet for 2 wk as follows: Group 1, 100%; Group 2, 75%; Group 3, 50%; and Group 4, 25%. Routine histologic evaluation was performed on longitudinal sections of paraffin-embedded hearts stained with hematoxylin and eosin. Computer-assisted image analysis was conducted on left ventricular free-wall of picrosirius red-stained sections for histomorphometric evaluation of ratio of cross-sectional area occupied by myofibers versus interstitium and for quantification of myofiber width. No differences were detected histologically among groups, and no difference in the mean myofiber: interstitium ratio was detected between Groups 1 (9.1) and 4 (9.5). Mean values for myofiber width ranged from 24.6 microns for Group 1 to 17.3 microns for Group 4. Two-way ANOVA revealed a strong effect of dietary restriction on reduction of myofiber width but no consistent gender effect. The significant dietary effects occurred in Groups 3 and 4 compared to corresponding controls. The present authors speculate that, if reductions in feed intake were < 50% in short-term preclinical studies, any reductions in myofiber width could imply a primary test article effect. Conversely, if reductions in feed intake were > or = 50% in such studies, reductions in myofiber width could be caused either solely by inadequate nutrition or by combined effects of nutrition and test article.