Accurate information on diet composition is central to understanding and conserving carnivore populations. Quantitative fatty acid signature analysis (QFASA) has emerged as a powerful tool for estimating the diets of predators, but ambiguities remain about the timeframe of QFASA estimates and the need to account for species-specific patterns of metabolism. We conducted a series of feeding experiments with four juvenile male brown bears (Ursus arctos) to (1) track the timing of changes in adipose tissue composition and QFASA diet estimates in response to a change in diet and (2) quantify the relationship between consumer and diet FA composition (i.e., determine “calibration coefficients”). Bears were fed three compositionally distinct diets for 90–120 days each. Two marine-based diets were intended to approximate the lipid content and composition of the wild diet of polar bears (U. maritimus). Bear adipose tissue composition changed quickly in the direction of the diet and showed evidence of stabilization after 60 days. During hibernation, FA profiles were initially stable but diet estimates after 10 weeks were sensitive to calibration coefficients. Calibration coefficients derived from the marine-based diets were broadly similar to each other and to published values from marine-fed mink (Mustela vison), which have been used as a model for free-ranging polar bears. For growing bears on a high-fat diet, the temporal window for QFASA estimates was 30–90 days. Although our results reinforce the importance of accurate calibration, the similarities across taxa and diets suggest it may be feasible to develop a generalized QFASA approach for mammalian carnivores.