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<i>Helichrysum italicum</i> is an aromatic plant with promising pharmacological activities. Bioactive compounds found in plants represent an important alternative treatment for weight loss and an infusion of <i>H. italicum</i> contains compounds which could have such effect. Our aim was, therefore, to investigate its acute effects on resting energy expenditure (REE) and possible differences in substrate oxidation in a pilot study. A dried, ground plant material of <i>H. italicum</i> ssp. <i>italicum</i> was infused with hot water and chemically characterized with HPLC-MS analysis. Sensory evaluation of herbal tea was performed. A randomized, crossover, controlled pilot study was then conducted on eleven healthy male subjects. The REE and substrate oxidation were measured by indirect calorimetry at baseline and 30 and 120 min after ingestion of infusion or hot water. The expression of genes involved in lipid metabolism was examined in <i>H. italicum</i> infusion-treated hepatocytes. Several phenolic classes were identified in the infusion, caffeoylquinic acids were the most abundant, followed by pyrones and flavonols. A single ingestion of <i>H. italicum</i> infusion significantly increased REE by 4% and fat oxidation by 12% compared to hot water ingestion. A significant 2-fold up-regulation of β-oxidation-related genes in HepG2 cells, exposed to <i>H. italicum</i> infusion, was detected. This pilot study suggests that <i>H. italicum</i> infusion possesses bioactive substances with potential application in obesity prevention, which could, with additional studies, become an economically interesting novel application of the plant. Clinical trial registration number: NCT04818905