*4.3. Supplement Formulation*

One 30-mL CPO drink contained 10 g of fish-derived collagen peptide (FUJIFILM, Tokyo, Japan), 400 mg of ornithine, and other ingredients including vitamin C, acidifier, and sweetener. To prepare fish-derived collagen peptide, gelatin was extracted from the fish scales, almost all from Tilapia, by hot water extraction. Gelatin was then digested by food-processing protease to prepare fish-derived collagen peptide. Total amount of hydroxyproline in fish-derived collagen peptide, which was analyzed by the hydrolysis of collagen peptide and subsequent high performance liquid chromatography, was 1100 mg in one CPO drink. The weight-average molecular weight of the collagen peptide was 2000 to 3000 Da. This drink contained less than 9 mg of potassium and 0.3 mg of magnesium. The placebo drink contained the same amount of vitamin C, acidifier, and sweetener, but did not contain CPO. One bottle was administered every day before bedtime for 8 weeks. On the test initiation day, the test drink was administered in the daytime because of the measurement of growth hormone levels. CPO and placebo drinks, including the bottle, were indistinguishable by shape, taste, or color. The dosage of collagen peptide was determined according to the previous report that showed the dose-dependent increase of blood hydroxyproline content by the intake of 2, 10 and 25 g of collagen peptide in humans [60]. In addition, the dose and the duration of this study were based on our previous study focusing on the effects of collagen peptide (UMIN: 000016587). The dosage of ornithine was based on an earlier study focusing on the effects of ornithine on sleep quality [42].

#### *4.4. Evaluation of Skin Condition*

For evaluation of skin conditions, we measured skin elasticity using a Cutometer MPA580 with a 6-mm diameter probe (Courage and Khazaka Electronic GmbH, Cologne, Germany). A 6-mm diameter probe was used, as opposed to a 2- or 4-mm diameter probe, to analyze functional changes in relatively deep areas of skin, such as the dermis. Furthermore, we analyzed skin elasticity at the neck because only thin muscles, such as the sternocleidomastoid muscle, are under the skin, making the neck a suitable area to analyze skin elasticity compared with the cheek, which mount on facial muscle [58]. To evaluate skin moisture, TEWL, and superficial pH at the cheek, a Corneometer (Courage and Khazaka Electronic GmbH, Cologne, Germany), VAPO SCAN AS-VT100RS (ASCH JAPAN Co., Tokyo, Japan), and Skin-pH-Meter PH905 (Courage & Khazaka Electronic GmbH, Cologne, Germany) were used, respectively. Skin conditions were also evaluated by VISIA Evolution (Canfield Scientific Ltd., Parsippany, NJ, USA), which analyzed spots, wrinkles, pores, texture, porphyrins, UV spots, red areas and brown spots from the picture of the participant's face [61], and DermaLab (Cortex Technology, Hadsund, Denmark), which analyzed echo-graphic density of subcutaneous tissue including epidermis, dermis and subcutaneous fat and calculated collagen score from its ultrasound image. All measurements of skin condition were evaluated in a testing room with stable temperature (21 ± 1 ◦C) and humidity (50 ± 5%).
