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In animal models, a role in the regulation of energy expenditure (EE) has been ascribed to sphingolipids, active components of cell membranes participating in cellular signaling. In humans, it is unknown whether sphingolipids have a role in the modulation of EE and, consequently, influence weight gain. The present study investigated the putative association of EE and weight gain with sphingolipid levels in the human skeletal muscle, a component of fat-free mass (the strongest determinant of EE), in adipose tissue and plasma.Twenty-four-hour EE, sleeping metabolic rate (SMR) and resting metabolic rate (RMR) were assessed in 35 healthy Native Americans of Southwestern heritage (24 male; 30.2 ± 7.73 years). Sphingolipid (ceramide, C; sphingomyelin, SM) concentrations were measured in skeletal muscle tissue, subcutaneous adipose tissue and plasma samples. After 6.68 years (0.26-12.4 years), follow-up weights were determined in 16 participants (4 females).Concentrations of C24:0, SM18:1/26:1 and SM18:0/24:1 in muscle were associated with 24-h EE (r = -0.47, P = 0.01), SMR (r = -0.59, P = 0.0008) and RMR (r = -0.44, P = 0.01), respectively. Certain muscle sphingomyelins also predicted weight gain (for example, SM18:1/23:1, r = 0.74, P = 0.004). For specific muscle sphingomyelins that correlated with weight gain and EE (SM18:1/23:0, SM18:1/23:1 and SMR, r = -0.51, r = -0.41, respectively, all P < 0.03; SM18:1/24:2 and RMR, r = -0.36, P = 0.03), associations could be reproduced with SMR in adipose tissue (all r < -0.46, all P < 0.04), though not in plasma.This study provides preliminary, novel evidence, that specific muscle and adipose tissue sphingolipid compounds are associated with EE and weight gain in Native Americans of Southwestern heritage. Further studies are warranted to investigate whether sphingolipids of different body compartments act in concert to modulate energy balance in humans.