Background: Human cardiac biopsies are widely used in clinical and fundamental research to decipher molecular events that characterize cardiac physiological and pathophysiological states. One of the main approaches relies on the analysis of semi-quantitative immunoblots that reveals alterations in protein expression levels occurring in diseased hearts. To maintain semiquantitative results, expression level of target proteins (TP) must be standardized. The expression of housekeeping proteins (HKP) is commonly used to this purpose. Methods and Results: We evaluated the stability of HKP expression (actin, b-tubulin, GAPDH, vinculin and calsequestrin [CSQ]) and total protein staining (TPS) within control (coefficient of variation) and comparatively with ischemic human heart biopsies (P value). All HKP exhibited a high level of intragroup (i.e., actin, b-tubulin and GAPDH) and/or intergroup variability (i.e., GAPDH, vinculin and CSQ). Among all, we found TPS to exhibit the highest degree of stability within and between groups, which makes of this reference the best to study protein expression level in human biopsies from ischemic hearts and age-matched controls. In addition, we illustrated that using an inappropriate reference protein marker misleads interpretation on SERCA2 and cMyBPC expression level after myocardial infarction. Conclusions: These emphasize the need to standardize the level of protein expression with TPS in comparative immunoblot studies on human samples from control and diseased hearts.