2.2.1. Target Population

The study was conducted between July to November 2019. Subjects were healthy men and women between the ages of 20 and 64 years who were presented with reduced motivation based on the vitality (VT) measured by 36-Item Short-Form Health Survey (SF-36) and poor quality of sleep based on the Pittsburgh Sleep Quality Index (PSQI) total score. The purpose and content of the study were thoroughly explained to the subjects both verbally and in writing, and the subjects consented to participation by providing written informed consent. This study was conducted in accordance with the tenets of the Declaration of Helsinki and the Ethical Guidelines for Medical and Health Research Involving Human Subjects. Protection of the human rights of the subjects was always ensured, and the study was implemented under the supervision of a doctor with the approval of the ethics review committee of the Miura Clinic (approval number: R1816, approval date: 27 June 2019). This trial is registered with UMIN ID: 000037726.

## 2.2.2. Test Method

This study was designed and implemented as a double-blind placebo-controlled trial. Subjects were selected after screening based on the level of motivation and quality of sleep. After baseline (week 0) measurements were obtained before the first dose, and the subjects received 500, 1000, or 3000 mg of *Euglena* powder or placebo powder (starch), divided into two doses per day (after breakfast and after dinner) for 12 weeks. The same measurements were taken at 4, 8, and 12 weeks after commencing intake. Subjects were instructed not to eat or drink after 10:00 p.m. on the day prior to the test and not to consume anything other than water until the designated meal was consumed on test day. On the

examination day, subjects consumed the designated meal (200 g of rice) at least 4 h before they visited the hospital and had only water until the end of the examination. On the day measurements were taken, the subjects underwent a subjective index survey using Visual Analog Scale (VAS), Profile of Mood States 2nd Edition (POMS2), SF-36, PSQI, Ogri-Shirakawa-Azumi Sleep Inventory MA Version (OSA-MS), autonomic system response indicator measurements, saliva collection, blood collection, and physical measurements such as height, weight and body mass index (BMI). After resting for 30 min, the subjects were subjected to the Uchida–Kraepelin [19–22] test for workload stress. The test took 30 min, including 15 min for the first half and 15 min for the second half with a break in the middle, while changing each line of simple single-digit addition once a minute. The correct answer rate for 15 min was calculated. The autonomic system response indicator measurements, saliva collection, and subjective index survey were repeated immediately after the Uchida–Kraepelin test. After resting for 60 min, the autonomic system response indicator measurements, saliva collection, and subjective index survey were performed again. During the test, the subjects drank water (about 100 mL) between the second subjective index survey and before starting the 60 min rest period.

#### Autonomic System Response Indicator Measurements

The balance of the autonomic nervous system, an indicator of fatigue and stress, was evaluated at three points for each test, i.e., before Uchida–Kraepelin stress loading, immediately after Uchida–Kraepelin stress loading, and 60 min after Uchida–Kraepelin stress loading. The tests were conducted before intake and at 4, 8, and 12 weeks after starting intake. The vital monitor VM-302 (Fatigue Science Laboratory Inc., Osaka, Japan) was used to simultaneously measure the pulse wave (PPG) and cardiac wave (ECG) signals, and to analyze high-frequency (HF) and low-frequency (LF) components of heart rate fluctuation [21]. HF reflects the parasympathetic response and LF reflects the sympathetic response.

#### Measurements of Health-Related Quality of Life through the 36-Item Short-Form Health Survey (SF-36)

The health of the subjects was surveyed with a quality of life questionnaire before intake and at 4, 8, and 12 weeks after starting intake using an SF-36 v2 [23–25], based on universally applicable health-related concepts. A five-point scale was used for responses to each of the 36 questions, and the questions included eight items; physical functioning (PF), role physical (RP), bodily pain (BP), general health (GH), vitality (VT), social functioning (SF), role emotional (RE), and mental health (MH).

#### Measurements of Mood States Using the Profile of Mood States 2nd Edition (POMS2)

Six items, anxiety and tension, hostility and anger, depression and discouragement, vitality and activity, fatigue and lethargy, and confusion and perplexity, were evaluated based on the T score using the Japanese version of the POMS2 [22,26,27] at three points for each test, before Uchida–Kraepelin stress loading, after Uchida–Kraepelin stress loading, and 60 min after Uchida–Kraepelin stress loading. Measurements were obtained before intake and at 4, 8, and 12 weeks after starting intake.

#### Visual Analog Scale (VAS)

The 100-mm VAS was used to quantify fatigue, irritability, concentration, mood, motivation, tension, and satisfaction with sleep for 1 week at three points for each test, before Uchida–Kraepelin stress loading, after Uchida–Kraepelin stress loading, and 60 min after Uchida–Kraepelin stress loading, before intake and at 4, 8, and 12 weeks after starting intake. Each VAS question had an endpoint of 0 indicating a "positive situation" and an endpoint of 100 indicating a "negative situation." Subjects were asked to mark the position on the line that represented their mood at the time. The distance from the endpoint of 0 to the mark was measured in mm.

#### Measurements of Quality of Sleep Using the Ogri-Shirakawa-Azumi Sleep Inventory MA Version (OSA-MA)

The sleep profile of the subjects was confirmed using OSA-MA [28,29], which includes five items related to sleep: sleepiness on rising, initiation and maintenance of sleep, frequent dreaming, refreshing, and sleep length. Items were assessed before intake and at 4, 8, and 12 weeks after starting intake. As for the score polarity, the direction of the good feeling of sleep had the highest score.

#### Pittsburgh Sleep Quality Index (PSQI)

The Japanese versionof the PSQI (PSQI-J) [29,30] was used to evaluate sleep quality before intake and at 4, 8, and 12 weeks after starting intake. The questions refer to the subject's sleep profile in the past month and can be assigned to seven components: subjective sleep quality, sleep latency, sleep duration, sleep efficiency, sleep disturbance, use of sleep medication, and daytime dysfunction. These seven categories are assigned scores from 0 to 3, and a higher total score (0 to 21) indicates a worse sleep quality.

#### Saliva and Blood Samples

Saliva samples were collected to measure cortisol, a stress hormone, at three points for each test, before Uchida–Kraepelin stress loading, after Uchida–Kraepelin stress loading, and 60 min after Uchida–Kraepelin stress loading, before intake and at 4, 8, and 12 weeks after starting intake. A blood test was also performed at these times to confirm the safety of ingesting the test substance before intake and at 12 weeks.
