*4.1. Study Design, Randomization and Blinding*

This study was a randomized, double-blind, placebo-controlled, parallel-group comparison trial to evaluate the effects of CPO dietary supplementation on skin conditions, plasma growth hormone and IGF-1 levels in healthy Japanese participants. Equal numbers of participants were allocated to active and placebo groups. This study, which was approved by the Kenshokai Ethical Review Board (Approved Number: 20170927-1) and registered in the UMIN Clinical Trials Registry (ID: UMIN000028924), followed the Declaration of Helsinki and Ethical Guidelines for Medical and Health Research Involving Human Subjects. Participants, clinicians, and practitioners were blinded. Practitioners performed intervention, outcome measurements, and analysis. Clinicians performed safety evaluation. According to our previous independent trial to evaluate skin TEWL or minimum erythema dose by dietary supplementation of astaxanthin in 10 healthy people [9,59], we set the required sample size as 10. The evaluation of skin elasticity was the primary outcome, while secondary outcome measures included other skin conditions such as skin moisture and TEWL, as well as plasma levels of growth hormone and IGF-1, and safety evaluation. Participants were enrolled and randomly allocated into the CPO or placebo group using a random number table with consideration of sex, age, skin elasticity, and moisture by practitioners. Allocation was concealed until all participants completed the tests.

#### *4.2. Participants*

Participants aged from 30 to less than 50 years in the Osaka area were included in this study. Every participant received an explanation of the objectives and details of this study, and provided written informed consent themselves. This study consisted of an 8-week ingestion period from October to December 2017. Participants meeting the following criteria were included in the study: (1) aged from 30 to 49 years at the time informed consent was provided; (2) exhibited relatively low levels of blood IGF-1; (3) exhibited relatively low skin moisture and skin elasticity; (4) BMI was less than 25; (5) capable of visiting the administrative facility on every inspection day; and (6) provided written informed consent for involvement in this trial themselves. Participants with the following criteria were excluded from the study: (1) continuous ingestion of a functional food or supplement; (2) continuous ingestion of a functional food or quasi-medicine with identical or similar effects as the active ingredient in test food; (3) frequent ingestion of food which rich in the same active ingredient in test food or ingestion of these kinds of food during the 3 days before and after trial initiation, or during the last 3 days of the trial; (4) worked a night shift or day and night shifts; (5) receiving medical treatment or prophylactic treatment, or diagnosed with the need for medical treatment; (6) presence of skin disease or abnormality in skin condition, such as atopic dermatitis; (7) exhibited apparent change of

skin condition that was not related to the intake of test food at the end of the trial compared with the initiation; (8) history of severe disease or abnormality in glucose metabolism, lipid metabolism, liver function, kidney function, or cardiovascular system function including heart, respiratory tract, endocrine system and nerve system function, or psychiatric disorder; (9) exhibiting anemia, or felt sick as a result of blood collection; (10) history of alcoholism or drug addiction; (11) risk of food allergy; (12) exhibited apparent abnormality in blood test or were positive for hepatitis B antigen or hepatitis C virus antibody in trial duration, including the screening period; (13) pregnant or lactating when informed consent was provided, or hoped to become pregnant during the trial; (14) involvement in another trial within 4 weeks prior to this trial, or participation in another concurrent trial; and (15) otherwise judged to be inappropriate for this trial by the clinician responsible for this trial.
