Context: Skeletal muscle and cardiac lipid accumulation are associated with diminished insulin sensitivity and cardiac function, respectively. In skeletal muscle, physical activity paradoxically increases fat accumulation, despite improvement in insulin sensitivity. Whether cardiac muscle responds similarly remains unknown. Objective: The objective of the study was to investigate cardiac lipid content and cardiac function after a 12-wk training program. Design: This was an intervention study with pre/postmeasurements. Setting: The study was conducted at Maastricht University Medical Center. Participants: Participants included 14 healthy, male overweight/obese subjects (age 58.4 ± 0.9 yr, body mass index 29.9 ± 0.01 kg/m2). Intervention: Intervention included a supervised 12-wk training program with three sessions per week (endurance and strength training). Main Outcome Measures: Maximal whole-body oxygen uptake, fasting plasma parameters, systolic function (by CINE-magnetic resonance imaging), and cardiac lipid content (by proton magnetic resonance spectroscopy) were measured. Results: Maximal whole-body oxygen uptake increased (from 2559 ± 131 to 2702 ± 124 ml/min after training, P=0.05). Plasma concentrations of glucose decreased (from 6.3 ± 0.2 to 5.7 ± 0.2 mmol/liter, P=0.001); plasma triacylglycerols and (free) fatty acids did not change. Also, body weight (from 94.2= 3.6 to 92.9 ± 3.6 kg, P = 0.10) and fat percentage (from 33.6 ± 1.7 to 32.5 ± 2.0%, P = 0.14) was unchanged. Left ventricular ejection fraction improved (from 52.2 ± 1.3 to 54.2 ± 1.2%, P=0.02), and cardiac lipid content in the septumwasdecreased after training (0.99 ± 0.15 to 0.54 ± 0.04%, P=0.02). Conclusions: Twelve weeks of endurance/strength training significantly reduced cardiac lipid content in overweight subjects and was paralleled by improved ejection fraction. This is in line with a lipotoxic action of (excess) cardiac lipids on cardiac function, although a causal relationship cannot be derived from this study. Further research is needed to clarify the clinical relevance of cardiac lipid content in the etiology of cardiovascular complications. Copyright © 2010 by The Endocrine Society.