Technical Abstract: High fat and copper deficient diets impair heart function leading to cardio hypertrophy, increased lipid droplet volume and compromised contractile function, resembling liptoxic cardiac dysfunction. However, the combined effect of the two on cardiac function is unknown. The purpose or objective of this study was to determine whether there is an interaction between high fat and copper deficient diets on cardiomyocyte contractile function. Weanling male rats were fed diets incorporating low or high fat diet (10% or 45% of kCal from fat, respectively) with adequate (6 mg/kg) or marginally deficient (1.5 mg/kg) copper content for 12 weeks. Contractile function was determined with an IonOptix system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), maximal velocity of shortening/relengthening (+/- dL/dt), intracellular Ca2+ rise and decay. Neither dietary treatment or intervention affected blood pressure or glucose levels although high fat diet elicited obesity and glucose intolerance. Both diets depressed PS, +/- dL/dt, increased intracellular Ca2+ rise, and prolonged TR90 and CA2+ decay, without additive effect between the two. Ca2+ sensitivity, apoptosis, lipid peroxidation, nitrosative damage, tissue ceramide and triglyceride levels were unaffected by either diet alone or in combination. Phospholamban but not SERCA2a was increased by both diets. eNOS was depressed with additive effect; i.e. interaction. Electron transport chain was unaffected although mitochondrial aconitase activity was inhibited by high fat diet. These data suggest that high fat and copper deficient diets impaired cardiomyocyte contractile function and intracellular Ca2+ homeostasis, possibly through a similar mechanism, without obvious lipotoxicity, nitrosative damage or apoptosis.