Twelve mice (five females and seven males, C57BL/6, The Jackson Laboratory, 6–17 months) were co-housed with their siblings. One animal was overweight at the start of the experiment (>45 g, body condition score 5), and was excluded; we thus show the data for 11 mice in Figure 1D. No animals showed signs of dehydration, or were dropped from the study. Each animal’s baseline weight was recorded on five consecutive days, while they had free access to plain water in their home-cage. We then switched their home-cage water to 2% CA, and recorded their daily weight. Laboratory shutdowns because of COVID-19 required us to cease this experiment, preventing a longer-term assessment of the effects of 2% CA water on body weight in older mice.

Mice maintain healthy weights and water intake on CA water. A, Average weight (as a fraction of each animal’s baseline weight) for 17 young animals, divided into four experimental groups. The age-matched weight curve expected for C57BL/6J mice (The Jackson Laboratory, 2015) on free water is shown as a reference (thick black line). On day 11 (the Friday of the second week after the intervention), the CA group was switched from 1% to 2% CA in their home-cage water bottle. Error bars show mean ± 68% confidence intervals across animals. B, Volume of water drank in the home-cage, estimated by measuring water bottles daily (days 11–29) for the cohorts of mice on free plain (blue) or 2% CA (red) water. Each data point is the average for one animal; boxplots show median and quartiles. C, Plain water consumed in 5 min of free access, measured on days 16, 24, and 29, indicated with gray shaded bars in A. Each data point is the average for one animal; boxplots show median and quartiles. D, Weight curves of 11 older animals, before and after switching from free plain water to 2% CA water. Age-matched expected growth curves for C57BL/6 mice (The Jackson Laboratory, 2018) are shown as a reference in black. See Extended Data Figure 1-1 for growth-corrected and individual weight curves.

Mice in this study were on average 44 weeks old on the first day of the experimental intervention. Expected weight curves were computed from weighings of age-matched animals not undergoing fluid restriction, from The Jackson Laboratory (2018).

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