Using doubly-labelled water to measure free-living energy expenditure: Some old things to remember and some new things to consider

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The doubly-labelled water (DLW) method provides the ability to measure the energy expenditure of free-living animals based only on the injection of two isotopes in water (one of oxygen and one of hydrogen) and traditionally the collection of 2 blood samples. We review here the fundamental basis of how the method works, and highlight how the choice of the appropriate calculation equation can have a large impact on the resultant estimates, particularly in species where the difference between the isotope elimination constants is small. This knowledge is not new, but is worth reiterating given the potential for error by making the wrong choice. In particular, it is important to remember that for mammals weighing less than 5kg, and birds weighing less than 2kg, the single pool models perform best in validation studies, while in mammals above 15kg the two-pool models perform best. Above 2kg in birds and between 5 and 15kg in mammals, however, the model superiority is uncertain. Even where the choice based on body mass would appear clear, the decision may need to be tempered by species specific information regarding potential additional sources for hydrogen turnover, such as de novo lipogenesis or methanogenesis. Recent advances in the technique have included attempts to make the method less invasive by using innovative methods for dosing and sample collection. In addition, the advent of laser spectroscopy, as a replacement technology for mass spectrometry, may open up many new opportunities in the field. These potentially include direct sampling of breath in the field and tracking background isotope drift using (17)oxygen levels.

Original languageEnglish
Pages (from-to)3-9
Number of pages7
JournalComparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology
Early online date1 Apr 2016
Publication statusPublished - Dec 2016

Bibliographical note

We are grateful to all our collaborators for their interesting discussions about their applications of the DLW method, and to Peter Thomson for the technical assistance. We are particularly grateful to Alexander Reik and Fuwen Wei, our collaborators on the pony and panda studies respectively. We also thank Elena Berman, Ed Melanson, Yosuke Yamada, Dale Schoeller, Herman Pontzer, Masaki Shirai, Michael Scantlebury, Klaas Westerterp and Guy Plasqui for their insights. Elena Berman kindly provided the plot in Fig. 1. Our own work with the DLW method has been funded by various grants from the UK NERC (NE/C004159/1) and BBSRC (BB/G009953/1, BB/J020028/1). John Speakman is currently a Royal Society Wolfson Professor and a ‘1000 Talents’ Professor of the Chinese Academy of Sciences.


  • doubly-labelled water
  • isotopes
  • dilution space
  • non-invasive


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