This dataset comprises characteristics of three-spined stickleback fish including length, weight, sex, condition factor (K-factor), cortisol and glucose concentration, RNA:DNA ratio and ethoxyresorufin-O-deethylase activity normalised to liver homogenate protein concentration. These data were collected by the Centre for Ecology & Hydrology from three-spined sticklebacks (Gasterosteus aculeatus L.) captured in the River Ray (south west England) at sites downstream of an urban waste water treatment works (Rodborne WWTW) prior to (2005-2007), and following (2008), remediation of the WWTW effluent with granular activated carbon (GAC) tertiary treatment. During the same period fish were also sampled from neighbouring reference rivers (R. Ock, Childrey Brook).