Abstract Background Obesity-mediated epicardial adipose tissue (EAT) expansion drives fat cell infiltration which forms the unique substrate for atrial fibrillation (AF). The LEGACY study showed the benefits of weight loss but an attenuated response with weight fluctuation. How fluxes in weight impacts the atrial substrate in not known. Objective To investigate EAT and the atrial substrates due to weight fluctuation, with comparison to stable obesity. Methods We studied 24 sheep in 3 equal groups over 80 weeks: 1. Obesity induced by high calorie diet fed ad libitum; 2. Weight fluctuation induced by 20-week cycle of weight gain/loss (20:20:20:20); and 3. Lean controls fed quality hay to maintain baseline weight. All sheep underwent: daily weight measurement; haemodynamic and imaging assessments (cardiac MRI; dual-energy X-ray absorptiometry; and matrix assisted laser desorption infrared lipid imaging); electrophysiological studies and electroanatomic mapping; histological and structural analysis. Evaluations included: atrial voltage, conduction velocity, and refractoriness (7 sites, 2 cycle lengths), electrogram fractionation, EAT volume, fibro-fatty infiltration, myolysis of myocytes, and spatial distribution of intra-atrial lipids. Results The Table shows the group differences. Compared to reference controls, obesity demonstrated: Increased atrial volume and pressure, abnormal atrial electrical properties, expanded EAT and ensuing fibro-fatty infiltrations, and myolysis of myocytes. Despite comparable weight and EAT with controls, weight fluctuation resulted in extensive and severe fibro-fatty infiltrations, and twofold greater myolysis that persisted. Moreover, characteristic profiles and abundance of lipid species in the atrial myocardium were noted on further evaluation. More importantly, EAT and fibro-fatty infiltrates strongly correlated with increased atrial volume and pressure; with only fibro-fatty infiltrates correlating with fractionated electrograms (r=0.71, p<0.001) and conduction slowing (r=−0.59, p=0.006). Similarly, atrial myolysis exhibited significant correlations with atrial enlargement and haemodynamics, and electrical substrates (p<0.05 for all). Conclusions Obesity induces fibro-fatty replacement of atrial myocytes and deterioration of contractile units, which may drive impaired electrical remodeling. Despite final weight loss, weight fluctuation demonstrates residual electro-structural, fibro-fatty and contractile substrates.