The target sample size was determined on the basis of a previous report of the anti-obesity effects of continued intake of PFE [12]. Using a significance level of 0.05 and statistical power of 0.80, the calculated sample size was 37 per group. Allowing for dropouts and withdrawals, the sample size was set at 40 participants per group (80 in total).

Volunteers were recruited with compensation, and participants were enrolled by the principal investigator in accordance with the inclusion and exclusion criteria. Before study initiation, all participants received a thorough explanation of the study and provided written informed consent.

Inclusion criteria were as follows: 1) healthy Japanese men aged 30 to 60 years at the time of consent; 2) BMI < 30; and 3) participants who received sufficient explanation of the study purpose and procedures, had decision-making capacity, voluntarily agreed to participate with full understanding, and provided written informed consent.

Participants were excluded if they: 1) had sleep disorders or chronic insomnia; 2) used medications that may affect obesity, hyperlipidemia, or lipid metabolism; 3) had a current or past history of thyroid dysfunction; 4) had a current or past history of serious hepatic, renal, gastrointestinal, cardiac, respiratory, endocrine, or other metabolic diseases; 5) had current otolaryngologic conditions or other diseases that preclude nasal breathing; 6) had a current or past history of drug or alcohol dependence; 7) had substantial body-weight fluctuations within the previous year (±5 kg or more); 8) had a habit of vigorous exercise; 9) had gastrointestinal diseases or a surgical history that affects digestion or absorption; 10) were unable to abstain from supplements or health foods, including Foods for Specified Health Uses and Foods with Function Claims, during the study period; 11) had self-reported food allergies; 12) had highly variable lifestyle habits or daily routines; 13) were following specific dietary restrictions or regimens, such as fasting, extreme carbohydrate restriction, or vegan diets; 14) were unable to abstain from alcohol from 2 days before screening and each assessment; 15) habitually consumed alcohol on 4 or more days per week in amounts exceeding 20 g/day of pure alcohol; 16) were smokers; 17) were shift workers or night-shift workers; 18) were judged by the principal investigator to be unsuitable on the basis of clinical examinations conducted at screening; 19) had donated more than 200 mL of blood within 1 month or more than 400 mL of blood, including component donation, within 3 months before consent; 20) were currently participating in, or intending to participate in, other studies involving consumption of foods, use of pharmaceuticals, or application of cosmetics; and 21) were judged as unsuitable for the current study by the principal investigator for other reasons.

This study was conducted under the oversight of a physician at the Nihonbashi Cardiology Clinic, Medical Corporation Foundation Kenko Suiminkai, in accordance with the Declaration of Helsinki (amended at the World Medical Association (WMA) Fortaleza General Assembly, Brazil, October 2013) and the Ethical Guidelines for Life Sciences and Medical Research Involving Human Subjects (partially revised March 27, 2023), following review and approval by the Ethical Committee of Kobuna Orthopedics Clinic (Chair: Toshio Kawada; approval date: December 18, 2025).

The study protocol was registered in the clinical trial registry system operated by the University Hospital Medical Information Network (UMIN) Center (Trial ID: UMIN000060312; title: Effects of Consumption of a Food Containing Plant Extracts on Resting Energy Metabolism - A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study).

This study was conducted from January to April 2026 as a randomized, double-blind, placebo-controlled, parallel-group comparative trial (allocation ratio 1:1) over a total of nine weeks, consisting of a one-week pre-observation period and an eight-week intake period. No methodological changes were made after study initiation.

Using computer-generated random numbers, the biostatistician performed block randomization (block size of four), adjusting for age, BMI, and estimated muscle mass. An allocation manager, who was not otherwise involved in the study, assigned the two randomized groups to the active group and the placebo group. The allocation manager prepared a table (key code) documenting the allocation results, sealed it, and kept it secured until after the analysis population had been determined, at which time the key code was unsealed. This procedure ensured blinding for all individuals other than the allocation manager.

To maintain blinding of both participants and intervention implementers, the study food was packaged in plain aluminum packets, with two tablets per packet, and distributed to participants. Throughout the study period, participants were instructed to refrain from using supplements and health foods, maintain their usual lifestyle as before study initiation, avoid excessive alcohol consumption, and refrain from participating in other trials. Participants were also instructed to consume the distributed standardized meal in the prescribed amount by 21:00 on the day before each comprehensive assessment; remain fasted from 12 hours before the measurements until all measurements were completed; limit fluid intake from waking until 1 hour before the assessment to no more than 200 mL of room-temperature water, with no further fluids consumed until completion of the measurements; avoid brisk walking and exercise on the assessment day; and avoid using stairs as much as possible. In addition, from 2 days before each comprehensive assessment, participants were instructed to abstain from alcohol and avoid strenuous exercise. From the day before each comprehensive assessment, they were instructed to refrain from physical labor and caffeine-containing beverages and to go to bed by around midnight to ensure sufficient sleep. Participants were required to use medications only with the approval of the principal investigator or a sub-investigator, except in emergencies.

During the intake period, participants consumed the study food as the intervention. The active food consisted of tablets manufactured by combining PFE (Toyo Shinyaku Co., Ltd.) with reduced maltose, cellulose, sucrose fatty acid ester, and silicon dioxide. For the placebo food, the PFE in the active food was replaced with caramel coloring, and the amount of reduced maltose was adjusted to render it indistinguishable in appearance from the active food. Both the active and placebo foods were designed to be taken at a daily dose of two tablets, each containing 250 mg. During the intake period, participants were instructed to consume one packet (two tablets) once daily with water or lukewarm water (the active group received the active food, and the placebo group received the placebo food). Table 1 shows the caloric content and nutritional composition per daily intake of the active food. The active food contained 35 mg of Pueraria flower-derived isoflavones (as tectorigenins) per daily intake, expressed as the sum of tectorigenin-7-O-xylosylglucoside, tectoridin, and tectorigenin.

Table 1. Analysis of nutrient composition values of study foods.

^aEnergy conversion factors: protein, 4; fat, 9; carbohydrate, 4; ^bNitrogen-to-protein conversion factor: 6.25.

The primary outcome was the resting fat oxidation, and the secondary outcomes were the resting carbohydrate oxidation, resting energy expenditure (REE), and respiratory exchange ratio (RER). Assessments were conducted twice: at baseline (pre-intake) and after 8 weeks of intake. No changes to outcomes were made after study initiation.

Both the primary and secondary outcomes were measured using the Aeromonitor AE-310S pulmonary exercise load monitoring system (Minato Medical Science Co., Ltd.). The resting fat oxidation, carbohydrate oxidation and REE were automatically calculated by the device using Elwyn’s equation for fat oxidation and carbohydrate oxidation and Weir’s equation for REE, respectively [20][21]. Assuming that protein oxidation was negligible, urinary nitrogen excretion (Nu) was set to zero in both equations.

On each assessment day, participants underwent a health status check after arrival, followed by measurements of blood pressure and body weight. Measurements were conducted in a room maintained at 23˚C - 25˚C. Participants remained seated at rest for 30 minutes while wearing a mask for expired-gas collection, then rested in the supine position for 30 minutes with data collected during the final 10 minutes after the first 20 minutes had elapsed. The gas analyzer and flow meter were calibrated before each measurement according to the device procedure, and proper values displayed on the device were confirmed after calibration. The first and last 1 minute of each measurement were excluded, and the mean values over the remaining stable 8 minutes were used to calculate each parameter following a previous study that used the same device [22].

Participants received a dietary diary and a participant diary and were instructed to record the following items daily from 1 week before the start of intake through the intake period: 1) meal start time, location, and meal contents; 2) study product intake status, during the intake period only; 3) presence or absence of any changes in physical condition; 4) sleep duration; 5) number of steps; 6) performance of any irregular exercise; 7) presence or absence of changes in lifestyle circumstances; and 8) use of medicinal products, excluding nutritional tonics and including newly designated quasi-drugs and newly classified quasi-drugs. In addition, from 1 week before the start of intake through the day before the 8-week post-intake assessment, daily physical activity was assessed using a pedometer. Dietary surveys were performed at baseline and on the day before the 8-week post-intake assessment using the Food Frequency Questionnaire (FFQ) Next, short form.

The analysis population was defined as the per-protocol set (PPS). Between-group comparisons of baseline values, week 8 post-intake values, and changes from baseline were conducted using unpaired t-tests. All tests were two-sided, with a significance level of 5%. Statistical analyses were performed using IBM SPSS Statistics 28 (IBM Corp., Armonk, NY, USA). Participant baseline characteristics are presented as mean ± standard deviation, and other data are presented as mean ± standard error. No additional analyses were performed.

Eighty participants were enrolled in this study. After randomization, no participants dropped out before the start of the study, and all 80 participants started the study, with 40 in each group receiving the allocated intervention. During the study period, one participant in the active group met a discontinuation criterion because he was unable to attend scheduled visits for reasons unrelated to the study. Consequently, 79 participants completed the study.

After study completion, 11 participants met exclusion criteria, leaving 68 participants for analysis. Reasons for exclusion were violation of study precautions during the intervention period (three participants in the active group and five in the placebo group) and substantial disruptions to lifestyle or dietary habits during the study period (two participants in the active group and one in the placebo group). Analyses were conducted for each group according to the original allocation.

Participant recruitment through completion of follow-up occurred from January 2026 to April 2026. Because follow-up was completed for all participants, the study was concluded. Baseline characteristics of participants included in the analysis set are presented in Table 2, and participant flow from enrollment to analysis is shown in Figure 1.

Figure 1.Flow diagram of progress through phases of randomized, double-blind, placebo-controlled, parallel-group study.

Table 2.Baseline characteristics of the participants.

Values are expressed as means ± SDs. No significant difference was observed.

Table 3.Resting energy metabolic parameters measured by indirect calorimetry.

Values are expressed as means ± SEs. (Placebo: n= 34, Active:n= 34); *Significantly different from the placebo group (P < 0.05).

The expired-gas measurement results are shown in Table 3. Resting fat oxidation increased from baseline to week 8 in the active group but decreased in the placebo group. No significant between-group difference was observed in post-intake measured values (P = 0.13). However, the change from baseline differed significantly between groups (P < 0.05). REE decreased from baseline to week 8 in both groups, but the measured REE value at week 8 was significantly higher in the active group than in the placebo group (P < 0.05). The change in REE from baseline also differed significantly between groups (P < 0.05). Carbohydrate oxidation decreased from baseline to week 8 in both groups; however, neither the measured values nor the changes from baseline differed significantly between groups (measured values, P = 0.16; changes from baseline, P = 0.64). Similarly, neither the measured RER values nor the changes in RER from baseline differed significantly between the placebo and active groups (measured values, P = 0.79; changes from baseline, P = 0.43).

During the study period, adverse events included common cold, nasal congestion, and eye fatigue in the placebo group and influenza in the active group. In all cases, the principal investigator determined that the adverse events were not causally related to the study foods. No serious adverse events were observed.