Nitrogen isotope analysis is a common technique for investigating dietary behaviour in modern and archaeological populations. One of its primary uses is to provide trophic level information. This application is possible because of a ∼3‰ enrichment in 15N along each step in the food chain, resulting in carnivores having higher δ15N values than herbivores, which in turn have higher δ15N values than plants. Much variation has also been observed within a trophic level, although the reasons for this are poorly understood. Here we present the results of a controlled feeding study designed to test the effects of gut anatomy and dietary protein levels on hair δ15N values within a trophic level. The data reveal that mammalian herbivores eating identical diets can have hair δ15N values that differ by as much as 3.6‰. This is particularly striking as it suggests that interspecific physiological differences can lead to larger shifts in δ15N values than a shift in trophic level. We also found that diet-hair fractionation was 2.3‰ greater when herbivores were fed high-protein (19%) diets than when they were fed low-protein (9%) diets. The primary nitrogen losses in mammalian herbivores are 15N-depleted urine and 15N-enriched faeces. We reason that an increase in the ratio of urinary to faecal nitrogen efflux leads to greater diet-hair fractionation on the high-protein diet. Copyright © 2003 John Wiley & Sons, Ltd.