Figure 1 Study Design and Behavioral Protocol (A) Schematic depiction of the overall study design. For full details, please see “Experimental Design” in the Supplemental Experimental Procedures. Pretest: subjects participated in a pretest, four exposure days, a posttest, and an fMRI scanning session. In the pretest, subjects were trained on how to use the scales to rate overall intensity, sweetness intensity, liking, and wanting for each of ten noncaloric flavored beverages developed in-house. After training, subjects rated each of the flavor stimuli three times. Average ratings were calculated, and two beverages were selected to be close to neutral in liking and equally well liked. One of the beverages was designated as destined to be the 112.5 calorie beverage, and the other was designated as the 0 calorie beverage. Caloric content was achieved by adding maltodextrin, which is relatively tasteless and odorless. To confirm that subjects could not detect the presence of the maltodextrin, we performed a triangle test with a separate flavored beverage (see B). Finally, subjects were trained on the fMRI procedures. Exposure sessions: subjects participated in four exposure days (two per stimulus). In each exposure day, subjects drank one of the beverages three times. Thus, each stimulus (0 calorie-paired flavored beverage and 112.5 calorie-paired flavored beverage) was exposed six times (three drinks × two days). Blood was sampled once for each beverage. Posttest: subjects rated all ten flavored beverages from the pretest (including the noncaloric version of the two exposed flavors) and filled out questionnaires. Subjects participated in one fMRI scan session within three weeks of beginning the experiment. The figure on the right depicts the setup in our mock scanner where they receive training in the procedure prior to scanning. During scanning, subjects sampled the noncaloric versions of the 0 calorie-paired flavor, the 112.5 calorie-paired flavor, as well as one of the nonexposed flavors as a control stimulus and a tasteless and odorless solution. (B) To verify that study participants were unable to detect the presence of maltodextrin, they participated in triangle tests in which they indicated which of three cups was different. All cups contained the same flavor, but for each trial, either one or two cups also contained maltodextrin. Eight trials were conducted. We used the binomial distribution to set our criteria for maltodextrin detection; specifically, the minimum number of correct judgments to establish significance for the triangle test (one-tailed, α = 0.05, z = 1.64, probability of guessing p = 1/3) was calculated according to the formula X=0.4714⋆z√n+[(2n+3)6], where n = number of trials, X = minimum number of correct judgments, and z = 1.64 if α is set to 5% [15]. For n = 8 tests, the minimum numbers of correct judgments is six. If they were unable to detect maltodextrin, they were given a lunch menu and asked to select items that would be provided as their lunch (sandwiches, chips, and fruit) in the exposure sessions. Each subject could select as many items as he or she wanted, but he or she was required to eat exactly the same thing for all lunch sessions. The number of correct trials for qualified subjects ranged from 2 to 5 (out of 8 possible), with an average of 3.58 and a SD of 1.16. Thus, no subject performed significantly differently from chance, allowing us to conclude that subjects were unable to detect the presence of maltodextrin in the flavored beverage. (C) Cartoon depicting the timing of stimulus delivery. There are two event types: “flavor” and “tasteless.” During flavor events, one of the three flavored beverages (control, 0 calorie-paired flavor, and 112.5 calorie-paired flavor) is delivered over 4 s. Note that no maltodextrin is added to the 112.5 calorie-paired flavor during scanning. Therefore, all flavors are noncaloric during scanning. Following delivery of the flavors, subjects are instructed to swallow and exhale through their nose so that the volatiles escape the oral cavity to reach the olfactory epithelium and induce retronasal olfaction, which is a key component of flavor perception. A variable period of rest (6–10.5 s) then follows. This “jitter” is important for aiding deconvolution of the hemodynamic response. Following the jitter, deionized water is delivered using the same procedure as the flavors, to rinse the mouth. During the tasteless event, the tasteless and odorless control solution is delivered over 4 s.