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Mitochondrial dysfunction and consequent cellular energetic failure play a key role in the development of sepsis-related organs failure. Evidence suggests that the pleiotropic effects of levosimendan may positively affect cellular metabolism during septic shock.To investigate changes in the concentration of glucose, lactate, pyruvate, and glycerol in the extracellular fluid of the skeletal muscle following levosimendan administration in patients with septic shock.The study was designed as a prospective, double-blind, controlled, clinical pilot trial and performed in a multidisciplinary intensive care unit. After achieving normovolemia and a mean arterial pressure of at least 65 mm Hg, 20 septic shock patients were randomized to receive either levosimendan 0.2 μg/kg/min (n = 10), or dobutamine 5 μg/kg/min as active comparator (n = 10). Interstitial tissue concentrations of lactate, pyruvate, glucose, and glycerol were obtained by using muscle microdialysis. All measurements, including data from right heart catheterization, were obtained at baseline and every 6 h for the following 72 h after randomization.The trial is registered with Clinicaltrials.gov, number NCT02963454.Compared with dobutamine, levosimendan increased interstitial tissue pyruvate concentration (153.3 ± 73 and 187. 2 ± 13.5 vs. 210.7 ± 76.2 and 161 ± 64.6; P < 0.05), and lactate clearance (55 vs. 10). Lactate/pyruvate ratio was lower in the levosimendan group at the end of study period (37. 7 ± 18.9 and 29.3 ± 12.7 vs. 10.9 ± 4.5 and 31.4 ± 13. 2; P < 0.05).Although we investigated a small number of patients, our preliminary results suggest that levosimendan may improve cellular metabolic alterations in patients with septic shock.