The porous structure and the state of the water are two main factors which define the vast applications of hydrogels in the life science arena. The structural characterisation and water state in hydrogels produced by the cryogelation of poly(hydroxyethyl methacrylate) and gelatine were undertaken using different techniques. Images obtained using confocal laser scanning and multiphoton microscopies were analysed using ImageJ/Fiji software to estimate the total porosity, specific surface area and pore size and wall thickness distribution functions of each of the hydrogels. The hydration properties and structural characteristics of the nanopore component of the polymer and protein hydrogels were analysed using DSC, 1HNMRspectroscopy and cryoporometry and modelled using the PM6 quantum chemical method. The hydrogels produced by cryogelation were shown to have a large macropore volume, high pore interconnectivity and small specific surface area. The main portion of water was shown to be attributable to bulk water located within macropores. The relative amounts of bound water in the hydrogels were demonstrated to be small (<10 wt% of bulk water) making macroporous hydrogels an attractive system for biological applications. An understanding of the parameters studied here is important for the future engineering of cryogels for biological applications.