Physiological responses after an acute handling stress and their subsequent effects on innate immune parameters, plasma free amino acids (AA) and liver energy substrates were assessed in Senegalese sole (Solea senegalensis). Eight groups of six specimens (136.1±58.4 g wet weight) were maintained undisturbed, while other eight groups of six specimens were used for acute stress challenge (air exposed during 3 min). A group of six specimens was sampled for blood and head-kidney collection immediately after air exposure (time 0), while the remaining groups were sampled at 5 and 30 min, 1, 2, 4, 6 and 24 h. Undisturbed fish were sampled at the same times and used as control. Fish were fasted for 24 h prior to air exposure and sampling. Plasma cortisol, glucose, lactate and osmolality levels increased immediately after stress peaking at 1 h in air exposed fish. Changes in plasma free AA were also observed at 1 and 24 h after stress. In liver, glycogen levels significantly decreased at 30 min and 1 h, while triglycerides values significantly increased at 1, 2 and 4 h in air exposed fish. In addition, total AA levels in liver augmented significantly at 2 h holding high until 24 h in air exposed specimens. The respiratory burst of head-kidney leucocytes from air exposed fish was significantly higher than that from control groups at 2 and 6 h after air exposure. On the other hand, plasma lysozyme activity significantly decreased at 4 h after acute stress in air exposed fish, while plasma alternative complement pathway followed an inverse linear relationship with respect to cortisol showing the lowest value at 1 h after air exposure. The present study suggests that Senegalese sole presents a stress response comparable to that observed in other teleosts. While some indispensable AA may be used for the synthesis of compounds related to the stress response or fatty acid transport, dispensable AA were probably mainly employed either as energy sources or in gluconeogenesis. Moreover, results from non-specific immune parameters assessed suggest that cortisol may act as regulator of the innate immune system.