This crossover, randomized, controlled intervention trial was conducted with healthy college-age male students of Hispanic ethnicity. Thirty individuals were voluntarily enrolled in the study after Research Ethics Approval. This research was carried out in accordance with The Code of Ethics (Declaration of Helsinki). Protocols were approved by the Ethics Review Board on Biomedical Research at the National University of Honduras (UNAH), IRB#00003070. Inclusion criteria were: young healthy males, between 18 - 30 years old, BMI: 20 - 25, free of chronic diseases, not consuming OTC or prescribed medications, non-smokers, with no soy or dairy allergies and who customarily consume breakfast. Participants attended an information session in which they completed a background assessments interview, and anthropometric measurements after informed consent. An initial weight assessment was conducted using an electronic digital scale SECA 803. Height was measured with a portable stadiometer SECA 213. Eligible participants (n = 24) completed the entire study and were included in data analysis protocols.

Subjects randomly consumed all treatments on different days. A three-day washout period between each treatment was required. On each intervention day, participants skipped breakfast and consumed each treatment supervised by Researchers to ensure compliance. Visual Analogue Scale (VAS) questionnaires were applied every hour post consumption to assess perceived sensation of satiety, hunger and appetite for a period of 4 hours. Participants were only allowed to drink water during the morning (6 - 11 am). An outline of the study design is shown in Figure 1.

Each subject consumed three treatments and one control beverage. During the intervention period, participants received 350 ml of each of the treatments and

the control. Beverages were consumed early in the morning each day in which evaluations were conducted. Beverages were produced in the Food Innovation Pilot Plant of Zamorano University following Good Manufacture Practices (GMPs). Each formulation was blended, homogenized, pasteurized and bottled in glass jars with the same equipment under the same processing conditions. The beverage’s viscosity were adjusted by using other ingredients in the formulation to reduce the effect of viscosity in the perception of satiety as previously suggested by Wanders et al. (2011) [19] . The control or blank (B) beverage contained 0 g of protein and 2 g of dietary fiber (DF); the High Protein (HP) beverage contained 30 g of protein and 2 g DF; the High Fiber (HF) beverage contained 11 g of DF and 0 g protein and a the HPHF beverage contained 30g protein and 11 g DF. A summarized nutrient composition of all treatments and the control is shown in Table 1. Nutritional composition for all beverages was determined using AOAC protocols. Corn soluble fiber (Fibersol®) and two soy protein isolates (Profam® and Clarisoy™) were provided by Archer Daniels Midland Co, Decatur IL. All beverages provided 238 ± 3.8 Kcal and showed a light smoothie type texture with a creamy appearance.

Subjective sensation of satiety, hunger and appetite were measured using a Visual Analog Scale (VAS). Subjects were asked to assess these parameters by repeated ratings using a 10-mm VAS scale anchored at either ends with the words for “not at all” and “Extremely”. The intensity, time and date of the measurements were recorded by participants using assessment sheets provided by the Researchers. Each day, measurements were taken pre-consumption, at 6:30 a.m., and post-consumption at 7:30, 8:30, 9:30, and 10:30 a.m. Lunch was provided and consumed ad libitum at 11:30 a.m. in the university cafeteria where a standardized menu is served daily to all students.

Food intake after lunch was calculated by weighing the meals pre- and post-consumption. Participants were instructed to consume as much food as desired until they were “comfortably full”. All participants received a tray with the same food and beverage options during lunch time. Food consumption data during lunch was analyzed using Food Processor Nutrition Analysis Software

(ESHA’s Food Processor®); total energy and macronutrient intake was assessed for each subject.

Treatments effect on satiety was measured by calculating scores for the sensation of satisfaction/fullness, hunger, and desire to eat (appetite) in the interval immediately after the consumption of beverages to immediately prior to lunch. Changes in satiety, hunger and appetite scores over time and changes in scores at each time-point were analyzed with a general linear mixed model for repeated measures using Analysis of Variance (ANOVA). Duncan’s Multiple Range Test (DMRT) post hoc was performed on all ANOVA measures. Energy intake and macronutrient consumption was quantified using the ESHA’s Food Processor LLC® software and the data was analyzed using one-way ANOVA. All data was reported as mean values and standard deviations or standard errors and statistical significance was accepted at p < 0.05. Statistical Analysis System (SAS®) version 9.4 was used for all analyses.

Thirty volunteers enrolled in the study but only 24 were considered for further analyses due to poor compliance. Participants were healthy young men free of chronic diseases or food allergies. All subjects confirmed they regularly ate breakfast. Subject’s average age was 22 ± 1.4 years and a mean body mass index (BMI) of 23.6 ± 1.6 kg/m². High compliance was observed due to good training procedures, effective and constant communication between researchers and subjects and careful supervision of beverage consumption protocols.

Our results indicate that all treatments had a significant effect on the perception of satiety at different points over a 4-hour period after consumption (p < 0.001). No significant differences in the perception of satiety between treatments and the control were observed in the first hour post-consumption (P = 0.56). This indicates that the bulky effect is confined to the first hour post-consumption (Figure 2).

After 2 hours the perceived sensation of satiety was significantly higher for HP, HF and HPHF compared to the control (B) and this effect was also observed after 3 hours of consumption (P = 0.02). After 4 hours, perception of satiety was significantly different only for HP (P = 0.02) compared to the control. This indicates the role of protein in promoting satiety and confirms similar finding by others [2] - [12] . Also these results indicate that beverages high in protein may have a prolonged satiety effect. Data in Figure 2 show how HP extended a full hour the perception of satiety when compared to a high carbohydrate beverage (control). This results are consistent with those of Marmonier et al., 1999, who

found that high-protein snacks provide a longer feeling of fullness than high-fiber and high-carbohydrates snacks [26] . Our data also supports the notion that subjective satiety and appetite perception should be assessed more than one hour post-consumption. Sunkin and Garrow (1982) were not successful in detecting differences when they evaluated satiety 60 minutes or less post-consumption [27] , whereas Porrini et al. (1997) having a longer assessment period (>60 min), had shown significant differences in satiety among macronutrients [28] . The return to hunger and food intake is usually better studied in a period of 3 - 7 h after treatments are consumed; studies taking measurements after 7 hours post-consumption have consistently failed to find differences in the perception of satiety or food intake [29] . Appetite perception was also measured to support our data on satiety. For all treatments, there was a significant time effect on the perception of appetite over the 4-hour period, with a marked reduction after 1 hour of consumption (p < 0.001). The major difference in appetite perception was observed after 2 hours where the perception of appetite for the high carbohydrate beverage was significantly higher compared to HP, HF and HPHF (P = 0.01). After 3 and 4 hours the results tend to follow the same behavior and were close to significance but not statistically different (Figure 3).

Hunger perception also changed throughout time in this study (P < 0.001) with an accentuated reduction during the first hour for all beverages. Contrary to the finding on perception of satiety and appetite, we didn’t observed significant differences among treatments at any point throughout the evaluation period (Figure 4). These findings are consistent with those of Cioffi et al. (2016) where differences in satiety and appetite were observed but no significance on hunger perception or subsequent food intake were observed between whole vs refined grain pasta meals [30] .

No synergistic satiety effect of fiber plus protein (HPHF) was observed in this study. Limited research has been conducted on the synergistic or additive effect

of fiber and protein on satiety. A study in adult dogs by Weber et al. (2007) concluded that a combined effect of a high fiber and high protein (HPHF) meal was more satiating and reduced the voluntary intake of food more than (HP) and (HF) meals alone [31] . We can speculate that data in animal models does not translate well into human populations as there are significant differences in consumption behavior.

High protein and high fiber meals have been evaluated separately and compared to each other in previous publications. Kranz et al. (2017) emphasized the importance of providing a high-quality nutritious breakfast to preschoolers that includes both protein and fiber, however he was unable to find significant differences in the feeling of fullness when comparing meals containing high protein or high fiber [32] . Rebello et al. (2014) concluded that protein or fiber sources have no effect on subsequent food intake. They evaluated several fiber and protein sources such as low-fat dairy products, eggs, legumes, oats, rye, and lupin and noted that while these foods seem to enhance the perception of satiety; this doesn’t always translate into a reduction in food intake [33] . This indicate how high fiber and high protein foods have a short term effects on appetite and hunger suppression, however, a deeper analysis need to be done on the right combination of satiety enhanced foods, their dose and timing of consumption to effectively asses subsequent food intake.

Although having a positive effect in the perception of satiety, the consumption of high protein, high fiber and high protein and fiber beverages did not resulted in a significant reduction in subsequent food intake compared to the a high carbohydrate beverage. Intake for Energy (812 - 870 Kcal), total fat (22 - 26 g), total carbohydrate (101 - 113 g) and total protein (41 - 50 g) showed a downward trend but non-significant differences were observed between treatments (P > 0.05) as illustrated in Figure 5. Probable increasing the sample size, the experimental groups or extending the consumption period may help to elucidate more significant differences among treatments. These limitations should be taken into consideration for future studies.

Our results are consistent with those of Cioffi et al. (2016) who concluded

that a high fiber food (whole grain) increased satiety, diminished hunger perception but did not decreased energy intake after a meal was consumed ad libitum [30] . Additionally, Vuholm et al. (2013) showed that sausages fortified with fiber ingredients significantly improved the satiety effect but no subsequent ad libitum energy intake was observed [34] .

Several publications have described how specific macronutrients, especially protein, have the ability to reduce food and energy intake. High protein meals (range 40 - 80 g/meal) were found to decreased energy intake (12% - 40%) in ad libitum meals 2 - 5 hours after consumption [28] [35] [36] [37] . Although such energy intake reduction was not observed in this trial, the effective satiety effect of high protein up to 4 hours and high fiber beverages up to 3 hours showcases the great potential to formulate products that might be effective as part of a weight management plan.

Considering the results and the timing of evaluation in this study, we conclude that beverages containing 30 g of plant protein or 11 g of soluble fiber may have the potential to control cravings and hunger feelings if consumed at mid-morning. This observation is consistent with the results obtained by Hull et al., (2015) in which subjects consuming a snack high in protein and fiber felt full for a longer period and had reduced hunger when consumed at mid-morning prior to an ad libitum meal [38] .