Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhoea in children and piglets. Infection of ETEC results in fluid secretion and electrolyte losses in the small intestine. In this study the effects of tempeh, a traditional fungal fermented soyabean product, on fluid losses induced by ETEC infection in piglets was investigated. Pairs of ETEC-infected and non-infected small intestinal segments of piglets were perfused simultaneously for 8 h with pre-digested tempeh, its supernatant and saline as an internal control. In saline perfused segments, ETEC infection reduced net fluid absorption by more than 500 μml/cm2, whereas this reduction was significantly less for pre-digested tempeh and its supernatant (75 and 282 μml/cm2, respectively). The supernatant of pre-digested tempeh was also compared with its permeate and retentate fractions. These fractions were created by ultra-filtration and contained respectively low and high molecular weight (>5kDa) compounds. Again ETEC infection caused a significant reduction of net fluid absorption when perfused with saline (386 μml/cm2) and also with the permeate fraction (300 μml/cm2), but much less with the supernatant and the retentate fraction (125 and 140 μml/cm2, respectively). The reduction in net fluid absorption upon ETEC infection when perfused with supernatant of either undigested or pre-digested tempeh was not different. Therefore from this study it can be concluded that a high molecular weight soluble fraction of tempeh is able to protect against fluid losses induced by ETEC, suggesting that this could play a potential role in controlling ETEC-induced diarrhoea.