Anticancer chemotherapy often induces side effects such as mucositis. Recent data suggest that a diet, Clinutren Protect (CP), containing whey proteins, glutamine, and transforming growth factor-beta (TGFbeta)-rich casein limits intestinal mucositis and improves recovery after a single methotrexate (MTX) challenge in rats. Chemotherapy consists of alternating periods of treatment and rest. Thus, our study evaluated the effects of CP on nutritional outcome and intestinal mucositis in rats receiving repeated chemotherapeutic challenges. Thirty-six Sprague-Dawley rats received 3 cycles of MTX at 8-d intervals. Rats had free access to CP or control diet (Co) from 7 d before the first MTX injection until the end of the experiment at d 27. In Co, whey proteins and TGFbeta-rich casein were replaced by TGFbeta-free casein. L-Glutamine was replaced by L-alanine. Body composition was assessed by dual energy X-ray absorptiometry. Before MTX challenges, food intake and body weight were similar in both groups but became higher during MTX challenges in CP (P < 0.05). Fat mass decreased similarly in both groups. In contrast, the decrease of fat free mass between d -1 and d 27 was less pronounced in the CP group (-9.5 g) than in the Co group (-57.2 g) (P < 0.05). The intestinal damage score was lower in the CP group (0.6 +/- 0.3 vs. 2.1 +/- 0.6; P < 0.05). Fecal IgA increased over time in the CP group (P < 0.05) but not in the Co group. A diet containing whey proteins, glutamine, and TGFbeta improves nutritional outcome by limiting the reduction of fat free mass and reduces intestinal mucositis during repeated chemotherapeutic challenges in rats.