Impacts of pre-sampling practices on fish plasma biochemistry may bias the outcome of a study if not considered within the general sampling strategy. Acute handling stresses can be imposed on fish during capture, and it is common practice to immobilise fish via sedation prior to obtaining blood samples for non-lethal extraction purposes, and/or to reduce stress, pain or suffering before being euthanised. We investigated these potential influences using a salmonid model (Oncorhynchus tshawytscha) by broadly measuring levels of 106 biochemical targets comprising ions, metabolites, and enzymes in samples of plasma. Two minutes of confinement with mild handling manipulation led to a significant departure from baseline metabolism, which was further exasperated during a prolonged five-minute challenge. These changes indicated disruption in osmoregulation, a switch towards anaerobic metabolism, and shifts in ammonia recycling, among others. Sedation of fish with clove oil and AQUI-S® had major impacts on plasma biochemical profiles, with alterations signalling changes in glycolytic metabolism, respiratory modes, carbon flux through the TCA cycle, and lipid compartmentalisation. Sedation was also characterised by enhanced levels of plasma amino acids, revealing a key difference between responses to handling stress and sedation. Targeted profiling also revealed that fish caught in large sea-pens via line and lure were less stressed than those captured via net crowding. These results demonstrate that pre-harvest practices should be carefully managed during fish sampling for biochemical/metabolomic-based analyses, and if manipulations are essential, they should be standardised.