Three anaesthetic agents were tested in sturgeon hybrid Acipenser naccarii female × Acipenser baerii male: clove oil at 100 mg L−1 (CLO); tricaine methanesulfonate at 150 mg L−1 (MS-222); combined medetomidine and ketamine hydrochloride (MK) administered by intravenous injection at 0.04 mg kg−1 + 4 mg kg−1, respectively. Efficacy of the anaesthetics was evaluated as well as physiological effects on blood gas status, acid-base balance and stress response by measuring blood PCO2, PO2, pH, inline image, haematocrit and serum cortisol, glucose, NEFA, lactate, Na+, K+, Cl concentration. Anaesthetic dosages were safe and effective in order to rapidly anaesthetize the sturgeons: induction time was significantly shorter with MS-222 (3.2 min) compared with CLO (4.7 min) and MK (5.4 min). Recovery time was significantly longer in MK anaesthetized fish (16 min) in comparison with CLO (5.8 min) and MS-222 (3.8 min). Anaesthesia induced blood hypercapnia, respiratory acidosis and stress response, with differences among anaesthetics. Blood PCO2 and cortisol levels were significantly higher in MS-222 (11 mmHg; 97.0 ng ml−1) and CLO (9.6 mmHg; 65.3 ng ml−1) anaesthetized groups compared to MK (7.8 mmHg; 39.6 ng ml−1) and control (7.0 mmHg; 10 ng ml−1). Overall results indicated that anaesthesia with MS-222 produced a greater physiological impact in sturgeons compared to the other anaesthetics. The effects were slightly attenuated in fish anaesthetized with clove oil, indicating that the use of this chemical was better than MS-222 in reducing handling stress. MK anaesthesia affected physiological parameters to a lesser extent. Intravascular administration of medetomidine at a dose of 0.04 mg kg−1 in combination with ketamine at a dose of 4 mg kg−1 proved to be a safe and effective anaesthetic protocol in this hybrid sturgeon, suitable for experimental studies and diagnostic procedures related to broodstock management and artificial reproduction.