Barley β-glucan (BG) has been shown to reduce glycaemic response (GR) in some studies. It is hypothesised that this reduction may be a function of its physical properties that delay gastric emptying (GE). The effect of these changes in GR and GE on diet-induced thermogenesis (DIT) is not known. The aim of the present study was to assess the effect of BG of different molecular weights and purities on GR, GE and DIT in healthy subjects. This was a randomised, single-blind, repeated-measures design where fifteen healthy subjects were tested on three occasions following an overnight fast. Following the baseline measurements, the volunteers were fed a soup containing high-molecular-weight BG (HBG), a soup containing low-molecular-weight BG (LBG) or a control soup with no BG (CHO). Following the consumption of the breakfast, GR was measured using finger-prick blood samples, GE was determined using the 13C-octanoic acid breath test and DIT was measured using indirect calorimetry. There was a difference in GR AUC between the soups after 60 min but not after 120 min. The CHO and LBG meals had a greater GR than the HBG meal. There were differences in all GE time points, with the HBG meal having the slowest GE time. There was a correlation between the GR and the initial GE times. There were differences in total DIT between the three test meals with the HBG meal having the lowest DIT. The present study indicates that HBG has the ability to delay GE due to increased viscosity, resulting in a decreased GR and DIT.