The athlete's paradox states that intramyocellular triglyceride accumulation associates with insulin resistance in sedentary but not in endurance-trained humans. Underlying mechanisms and the role of muscle lipid distribution and composition on glucose metabolism remain unclear. We compared highly trained athletes (ATHL) with sedentary normal weight (LEAN) and overweight-to-obese (OVWE) male and female individuals. This observational study found that ATHL show higher insulin sensitivity, muscle mitochondrial content, and capacity, but lower activation of novel protein kinase C (nPKC) isoforms, despite higher diacylglycerol concentrations. Notably, sedentary but insulin sensitive OVWE feature lower plasma membrane-to-mitochondria