Various “control animals” used in studies on biotin-deficient rats have been evaluated. Biotin-deficient and control rats fed ad libitum, and control animals given restricted amounts of food (10 g) as one, two or four equal meals a day were used. Average food intake of control and deficient animals fed the basal diet ad libitum were 17.1 and 9.5 g, respectively. Control animals given a restricted amount of food (10 g) as one, two or four equal meals a day consumed each allotment in 6 hours, 30 and 15 minutes, respectively. The rate of food ingestion appeared to be higher in animals given multiple meals than in animals given a single meal. Systematic oscillations in body weight, liver weight, the level of hepatic glycogen and spontaneous activity, were observed in control animals given a single meal. Furthermore, these oscillations decreased proportionately in animals given multiple meals. Similar oscillations were also observed in body weights of pair-fed animals. A threefold increase in spontaneous activity was observed in animals given a restricted amount of food as a single meal compared with animals fed ad libitum. This activity increased with progress of the fast. In view of these results it is apparent that “pair-fed,” “pair-weighed” and “trained-fed” control animals are different from biotin-deficient and control animals fed ad libitum.