*3.3. <sup>E</sup>*ffi*cacy Evaluation*

#### 3.3.1. Autonomic System Response Indicator Measurements

The low-frequency/high-frequency (LF/HF) ratio is summarized in Table 2. LF and HF, which represent the balance of the sympathetic nervous system and the parasympathetic nervous system in the autonomic nervous system, were measured 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. The LF/HF ratio was 3.7 in the placebo intake group, 3.3 in the *Euglena* 500 mg intake group, 4.1 in the 1000 mg intake group, and 4.7 in the *Euglena* 3000 mg intake group after Uchida–Kraepelin stress loading at week 0. The LF/HF ratio was 6.3 in the placebo intake group, 4.7 in the *Euglena* 500 mg intake group, 2.9 in the 1000 mg intake group, and 5.1 in the *Euglena* 3000 mg intake group after Uchida–Kraepelin stress loading at week 4 of intake. These findings sugges<sup>t</sup> that the continuous intake of *Euglena* reduced disruptions in the balance of the autonomic nervous system. However, there were no significant differences.

The mean change amount of the LF/HF ratio before and after Uchida–Kraepelin loading at week 0 was 0.7. Thus, we defined subjects with a change amount greater than 0.7 as being more prone to workload stress based on Uchida–Kraepelin (Table 3). The LF/HF ratio in the *Euglena* 1000 mg intake group after Uchida–Kraepelin stress loading at week 4 of intake was significantly lower than that in the placebo intake group (*p* = 0.024, Dunnett's test). This suggests that the intake of *Euglena* 1000 mg regulates the balance of the autonomic nervous system during work stress. No difference was found in the balance of the autonomic nervous system in subjects without Uchida–Kraepelin stress. After week 8 of intake, these differences were difficult to discern.


**Table 1.** Baseline data for subjects. After accounting for normality, the baseline characteristics of the participants were analyzed by one-way ANOVA or Kruskal–Wallis test.

BMI: body mass index, SF-36: 36-Item Short-Form Health Survey, VT: vitality, LF/HF: low-frequency/high-frequency, PSQI: Pittsburgh Sleep Quality Index.

*p*-value (Kruskal–Wallis test) - - - - - - - - 0.556


**Table 2.** Summary of LF/HF ratios, an indicator of autonomic nervous system balance. Low frequency (LF) and high frequency (HF) indicate sympathetic and parasympathetic nervous, respectively. After accounting for normality, a two-way repeated measure ANOVA was performed. The results were not significantly different.

LF/HF: low-frequency/high-frequency.

**Table 3.** Summary of LF/HF ratios, an indicator of autonomic nervous system balance (Subjects defined as susceptible to stress). After accounting for normality, a two-way repeated measure ANOVA was performed. The results showed a session x group interaction in "After" (*p* = 0.036) and were followed up with a post-hoc comparison using Dunnett's test. Dunnett's test was used for intergroup comparisons of the means of the placebo group and *Euglena* intake groups before and after Uchida–Kraepelin stress loading, and 60 min after loading at 0, 4, 8, and 12 weeks.


\* *p* < 0.05 vs. the placebo intake group. LF/HF: low-frequency/high-frequency.

#### 3.3.2. Analysis of SF-36

Table 4 summarizes the SF-36 results. The role physical (RP) values were significantly higher at weeks 8 and 12 of treatment with *Euglena* 3000 mg intake group (53.5 ± 5.2 and 55.4 ± 3.5) than in the placebo intake group (47.3 ± 8.4 and 49.2 ± 8.2), suggesting that intake of *Euglena* improves RP (*p* = 0.036, *p* = 0.021, Dunnett's test). At week 12 of intake, vitality (VT) tended to be higher in the 3000 mg *Euglena* intake group (51.2 ± 7.2) than in the placebo group (45.3 ± 5.5), indicating that the continuous intake of *Euglena* may also improve vitality (*p* = 0.065, Dunnett's test).

**Table 4.** Analyses of role physical (RP) and vitality (VT) based on SF-36. After accounting for normality, a two-way repeated measure ANOVA was performed. The results showed a main effect of group in RP (*p* = 0.023) and a session x group interaction in VT (*p* = 0.052). Therefore, we followed up with post-hoc comparisons using Dunnett's test. Dunnett's test was used for intergroup comparisons of mean values in the placebo intake group and *Euglena* intake group at 0, 4, 8, and 12 weeks.


\* *p* < 0.05, † *p* < 0.1 vs. the placebo intake group. SF-36: 36-Item Short-Form Health Survey, RP: role physical, VT: vitality.
