These data were collected from three-spined sticklebacks (Gasterosteus aculeatus L.) sampled at sites downstream of wastewater treatment works (WWTWs), and at sites with no identifiable WWTW input, in the north west of England. For fish captured at each site, data include fish characteristics (weight, length, condition factor and sex). Additionally, indicators of stress (whole-body concentrations of cortisol, cortisol released to water, and whole-body glucose concentrations) were quantified in order to assess the status of the stress axis in individuals immediately following capture (unstressed - baseline cortisol and glucose) and after a short period of confinement (stressed - elevated cortisol and glucose). The data were collected as part of a study conducted to evaluate whether the complex chemical milieu present in rivers downstream of WWTWs, one of the most abundant point-sources of aquatic pollutants in UK waters, can affect the functioning of the stress axis in fish. Sampling was conducted during 2011, 2013 and 2014 by the Centre for Ecology & Hydrology. Full details about this dataset can be found at https://doi.org/10.5285/87281ce8-6fb3-40c8-89de-0c476d84b110