We analyzed mice of different ages to investigate the factors that contribute to the disturbance of water balance with aging, and found that insensible water loss increases with age, contributing to the dehydration that occurs when water intake is reduced. The two major routes of insensible water loss are diffusion through skin and evaporation from the respiratory tract, but the effects of aging on them have not been extensively studied.
One route of insensible water loss is passive diffusion of water vapor through the barrier of the skin (Trans-Epidermal Water Loss, TEWL). Instrumentation developed to measure TEWL includes both open and closed chamber devices 
. Although deterioration of the skin with age increases its susceptibility to injury and slows repair of its barrier function, basal TEWL was found to be unchanged, or even decreased, with aging both in humans and mice (
and reviewed in 
). Nevertheless, older mice are larger, so there could be more evaporation through the greater area of skin. While the mice were in water balance, eating gel food with 56% water content, the older mice excreted 42±5% less urine, while their calculated body surface area is 25±5% greater. Considering that the older mice also had less water in their feces, the greater skin surface area, without any change in water permeability of the skin, apparently accounts for only part of the difference in insensible water loss.
Increased respiratory evaporation could also contribute to increased insensible water loss during aging. Aged normal lungs have dilated alveoli, enlarged airspaces, decreased exchange surface area and loss of supporting tissue in the peripheral airways 
. However, in the absence of direct measurements of respiratory water evaporation we cannot evaluate its possible contribution to aging related dehydration. These limitations call attention to the need for additional studies using novel methods if we are to understand the basis for the age related increase of insensible water loss.
We found that the older mice, but not the youngest ones, increased excretion of AVP in their urine when their intake of water was reduced (). Despite numerous studies of the effect of aging on plasma AVP in various animal species, including humans, it has still is not been clear how aging affects the level of AVP. The results are conflicting. While some studies report increased basal plasma AVP with aging, others do not, and some even report a decrease (reviewed in 
). In our experiments excretion of AVP was equal in mice of all ages while they were in water balance, consuming equal amounts of gel food containing 56% water (). However, when water was restricted, the older, but not youngest, mice increased their excretion of AVP, as they went into negative water balance due to increased insensible water loss. Thus, differences in hydration might have contributed to variability of the findings concerning the effect of aging on the level of AVP.
Summary and conclusions: 1) While consuming gel food containing 56% water, mice of all ages consume the same amount of food and water and maintain water balance, but the older mice excrete less urine of higher osmolality and excrete drier feces (). Thus, older mice have increased insensible water loss. 2) When water is restricted, older mice compensate the increased insensible water loss by increasing AVP excretion, accompanied by further concentration of their urine and lower water content of their feces. 3) The higher AVP that maintains hydration in older mice could contribute to age related pathology 
. 4) Young mice require less water to maintain hydration (). 5) Urine concentrating ability of 27 month old mice is less than that of the 3 and 18 month old mice, as seen when water intake is lowest (). 6) Increased insensible water loss occurs at a younger age (18 months) than decrease of urine concentrating ability (27 months).