**Average Daily Percent Bodyweight Changes**

**Figure 3.** Values are average daily bodyweight percent changes in the intervention and in the control groups; *n* = 46. \* indicates statistical significance (*p* < 0.05). The lines within the individual data points represent each group's mean and standard deviation.

## **4. Discussion**

These results sugges<sup>t</sup> that the INT group was able to improve their hydration status based on the decreased Uosmo and urine color from weekly laboratory visits. Although there was an observed decrease of dehydration in the INT group, both groups had clinical signs of dehydration from the spot samples provided (Uosmo > 800mOsm·kg−1). This would sugges<sup>t</sup> that although the INT groups did see improvements to their Uosmo, the INT condition was not sufficient to attain clinical euhydration. Despite being different at a statistically significant level between groups, urine color during the lab visits was similar for both groups, having a value of ~5 and a mean difference of only 0.5. Furthermore, USG and thirst perception during the lab visits were not significantly different between groups, suggesting that having accessibility to the Creative Roots® beverage may only improve Uosmo. However, the improvements in Uosmo may sugges<sup>t</sup> that the Creative Roots® beverage offers a potential benefit for hydration, due to the validity of Uosmo for assessing hydration [15]. The at-home morning urine color and morning and afternoon thirst perception did not show any change in the INT group when the participants were self-assessing their thirst perception and urine color.

Our results are similar to those of Khan et al. [1] who examined hydration markers during a 4-day water intake intervention in children with a prescribed low intake, high intake, and ad libitum intake of fluid. The authors reported Uosmo and urine color values in their ad libitum group that would approach the threshold for clinical dehydration, similar to what we observed in our participants, who were able to consume fluid ad libitum [1]. This similarity may sugges<sup>t</sup> that children with an ad libitum beverage intake may not see improvements in hydration and that a prescribed beverage intake may be more beneficial for improving hydration.

Interestingly, the at-home data (i.e., morning and afternoon urine color, bodyweight, and thirst perception) showed contradictory results. First, the afternoon urine color was lighter for the INT group; however, due to the low reported mean difference, we speculate that it is unlikely that these results have any clinical significance. Additionally, daily percent bodyweight changes were found to be statistically significant between the groups. The intervention group presented greater differences in daily percent bodyweight changes when compared to the control group, suggesting the intervention group was less hydrated than the control group. Bodyweight changes have been noted as one of the more practical markers for hydration, whereas day-to-day bodyweight losses of more than 1% can be an indicator of dehydration [16]. Although the intervention group did not lose over 1% of bodyweight, the group still lost more bodyweight than the control group, which is contradictory with their afternoon urine color measures. However, the bodyweight changes may be related to a reduced consumption of sugary beverages, thus a reduced caloric intake, due to having access to Creative Roots®. Prior research has demonstrated that reducing sugary beverage intake and replacing these beverages with non-caloric ones can result in reduced weight gain [17,18].

Contrary to previous research, we did not find any differences in mood between INT and CON, using the POMS-a. Fadda et al. [6] found that serving children 300–500 mL of water during the school day can improve their mood. This was a prescribed amount, whereas in the present study we utilized ad libitum fluid consumption and only assessed mood in children once per week, which may explain why we did not find any differences between our groups. This may sugges<sup>t</sup> that acute changes in hydration may impact mood, but it is possible that those differences are not observable when only assessed one time per week. We also did not observe any differences in sleep quality, using the PSQI, between the intervention and the control groups despite the intervention group having a lighter urine color in the afternoon. This finding may simply be due to the intervention not changing the hydration status. Further research is warranted, as there is little evidence available regarding hydration status and sleep quality in children.

## **5. Limitations**

There are several limitations to the present study. First, the intervention group was not prescribed a specific amount of fluid. We required the participants to have drinks available and accessible at each meal (i.e., breakfast, lunch, and dinner), as well as ad libitum throughout the day, but did not require them to consume specific amounts. Second, we did not ask the children or their parents to record what was consumed throughout each week. We also used first morning urine samples for hydration analysis (i.e., urine color, USG, Uosmo) in all lab visits in order to reduce scheduling conflicts with the participants and their caretakers. Recent literature suggests that Uosmo of afternoon urine samples is more representative of values observed in 24 h urine collection [14]. Lastly, the daily log data collection was not supervised by the research staff, though research participants were provided detailed instructions to promote compliance.

## **6. Conclusions**

Our results indicate that having access to Creative Roots® seems to produce a small but potentially meaningful benefit in hydration, as indicated by Uosmo and urine color. However, even with the observed improvements, children were still consistently mildly dehydrated regardless of the group. Our data show the intervention group did improve some biomarkers of hydration, but no effect was observed in at-home measures, mood, or sleep. Further research is warranted using a prescribed amount of fluid to determine if the

beverage improves the hydration status and monitoring dietary and fluid intake during the entirety of the study.

**Author Contributions:** Conceptualization, M.R.S., G.E.W.G., M.C.M., C.L.B., Y.S., R.L.S. and D.J.C.; methodology, M.R.S., G.E.W.G., M.C.M., C.L.B., Y.S., R.L.S. and D.J.C.; investigation, M.R.S., G.E.W.G., M.C.M., C.L.B., Y.S., C.N.M. and R.L.S.; data curation, M.R.S., G.E.W.G., M.C.M., C.L.B., Y.S., C.N.M. and R.L.S.; writing—original draft preparation, M.R.S. and G.E.W.G.; writing—review and editing, M.R.S., G.E.W.G., M.C.M., C.L.B., Y.S., C.N.M., R.L.S. and D.J.C.; project administration, D.J.C.; funding acquisition, D.J.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Kraft Heinz Company (Chicago, IL, USA).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the University of Connecticut (H19-212).

**Informed Consent Statement:** Participants provided verbal assent and their parents or guardians provided written consent prior to participation.

**Data Availability Statement:** Data available only on request due to ethical restrictions.

**Acknowledgments:** We would like to thank Fatou Lack, Mark Garcia, Michaela Pruchniki, Jonathan Granata, Radha Patel, Jeb Struder, Erica Filep, Erin Dierickx, and Rachel Katch for their assistance with data collection.

**Conflicts of Interest:** The authors declare no potential conflict of interest.
