Acute exposure to nicotinic agonists induces myotoxicity in zebrafish embryos. The main goal of this work was to evaluate the potential myotoxicity of nicotine acetylcholine receptor agonists on adult zebrafish muscle tissue by using nicotine as a model compound. Liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) datasets were processed with different chemometric tools based on the selection of Regions of Interest (ROI) and Multivariate Curve-Resolution (ROI-MCR procedure) Alternating Least Squares (ALS) for the analysis of different exposure experiments. Analysis of Variance Simultaneous Component Analysis (ASCA) of changes on metabolite peak profile areas showed significant nicotine concentration and exposure time-dependent changes, clearly differentiating between exposed and non-exposed samples and between short (2 h) and long exposure times (6 h or 24 h). Most of the changes observed in the concentrations of different metabolites are probably secondary to the observed hyperlocomotion, as they have been also observed in humans after strenuous muscular exercise. The absence of myotoxicity might be related with the reduced calcium permeability of adult muscle-type nicotinic acetylcholine receptors (nAChRs).