Doubly labelled water

The doubly labelled water (DLW) technique measures total carbon dioxide production by observing the differential rates of elimination of a bolus dose of the stable isotope tracers, 2H (deuterium) and 18O. Combined with an estimate of the respiratory quotient, this yields an estimate of total energy expenditure. The theoretical considerations and assumptions which underpin the method are complex and comprehensive reviews of these are listed in the reference section. 

DLW is the gold standard method for assessing total energy expenditure in free-living individuals. The method is unable to provide any information about the frequency, duration or intensity of bouts of activity, or energy expenditure occurring on a daily basis. The method is also unable to record the type or context of activity. The choice of DLW to measure physical activity or as a criterion method in a validation study is therefore limited to questions relating to overall energy expenditure over a number of days. It may be combined with a measure of resting energy expenditure to enable calculation of physical activity energy expenditure. The dimensions of physical activity assessed by DLW are described in Table P.3.21.

Table P.3.21 The dimensions of physical activity which can be assessed by doubly labelled water.

Dimension Possible to assess?
Total physical activity energy expenditure
Timing of bouts of activity
Contextual information (e.g. location)
Sedentary behaviour

The DLW method was originally developed by Lifson et al (1955) with many refinements since. Schoeller (1983) was the first to use the method in humans when the cost of 18O reduced sufficiently to make this a viable albeit expensive measurement for research. In subsequent years advances in measurement technology and the falling cost of the isotopes has made the method more accessible and it is now used routinely.

The method has been used in adults, children and infants to measure total energy expenditure (TEE), in many diverse investigations including the energy expenditure of clinical populations, and the energy utilisation of people participating in intensive physical activities under extreme conditions.

It has also been used widely in validation studies of methods of assessment of physical activity. DLW is also used for dietary assessment, as reported dietary intake should equal measured total energy expenditure in weight stable participants. Indeed, the application of DLW led to seminal work in the identification of widespread under-reporting in dietary assessment.

In physical activity measurement, DLW has been used to validate various methods which estimate energy expenditure, e.g. questionnaires, diaries, logs and body-worn sensors; the combination of DLW and resting energy expenditure measured using indirect calorimetry provides a robust method of measuring the energy expenditure due to physical activity.

Estimating total energy expenditure

Estimates of CO2 production are converted to estimates of total energy expenditure using food composition data and a modified Weir equation (Weir, 1949; Speakman, 1998).

The nature of the metabolic fuel being used plays an important role in the quantity of CO2 liberated, relative to the quantity of oxygen consumed. This relationship, which is characterised by the respiratory quotient (RQ) must be taken into account when deducing total energy expenditure from DLW experiments.  Approximate expressions which assume normal ratios of fat, carbohydrate and protein being metabolised have been derived but more accurate estimates can be obtained from the analysis of food diaries to derive individual RQ values.

Average total daily energy expenditure (TDEE) can be calculated by dividing by the duration of measurement in days.

Estimating physical activity energy expenditure

Estimates of physical activity energy expenditure (PAEE) are made following measurement of the other components of total daily energy expenditure. Resting energy expenditure (REE) can be assessed using indirect calorimetry or prediction equations (Frankenfield et al., 2005). Thermic effect of eating (TEF) can also be estimated using dietary records or estimated as approx. 10% of TDEE (Westerterp, 2004). The following equation is used:

PAEE (kcal/day) = TDEE (kcal/day) − REE (kcal/day) – TEF (kcal/day)

Physical activity level

Physical activity level (PAL) can be calculated using the ratio of average total daily energy expenditure to resting energy expenditure. This provides an index of physical activity which typically ranges from 1.40 to ~2.40 in adults (Hills et al., 2014).

In practice the participant is asked to drink a known dose of water enriched in 2H and 18O. The tracers used are non-radioactive and occur naturally in all water sources (including drinking water), and therefore completely safe to use in any population.

The fundamental basis of the DLW technique is that whilst the hydrogen label is lost only as water, the oxygen label is lost as both water and carbon dioxide; transference of oxygen between water and carbon dioxide is the consequence of rapid exchange promoted by carbonic anhydrase. Therefore, the difference between the turnovers of the two labels is a measure of the production of carbon dioxide. 

Samples of blood, saliva or urine, from which the isotopic composition of the body water can be determined, are collected over the next 5 – 14 days. From the isotope disappearance curves four parameters are deduced, the two pool sizes of hydrogen and oxygen and the fractional rate constants of elimination for each of these species, and these are combined to give an estimate of CO2 production.

 Key instructions for participants

  1. A baseline urine sample is vital to the measurement. If a participant forgets to bring a sample, another must be collected prior to dosing.
  2. Participants should be supplied with urine containers which are labelled day 1 to day 14 with the date and a space for the time to be recorded.
  3. Participants should also record the date and time of the urine samples on a log sheet.
  4. Participants should be instructed to collect a urine sample every day for 14 days after dosing and place into the fridge. Freezing is not required by participant.
  5. Urine should not be collected from the first void in the morning.
  6. Participants should not completely fill the container with urine as it will expand when the sample is frozen.
  7. Participants should be reassured that if they forget one urine sample to carry on to day 14 regardless.
  8. Ensure the urine samples are collected and frozen immediately after the collection period ends.
  9. Participants must record any episodes of vomiting or diarrhoea.

An overview of the characteristics of DLW is outlined in Table P.3.22.


  1. Gold standard method for measuring total energy expenditure in free-living participants.
  2. Does not interfere in participant’s daily activity.
  3. Relatively low participant burden.
  4. No feedback for participant reducing reactivity.
  5. Provides criterion validity for estimates of total energy expenditure and reported energy intake.
  6. Easy to administer in all populations including during pregnancy, infancy, old age.
  7. Also provides a measure of total body water, and hence body composition.


  1. The method is expensive due to the price of 18
  2. Mass spectrometry is also expensive.
  3. Does not give a direct measure of energy expended in physical activity.
  4. Does not give any indication of the intensity, frequency, duration or domain of physical activity.

Table P.3.22 Characteristics of the doubly labelled water method.

Consideration Comment
Number of participants Small
Relative cost High
Participant burden Low
Researcher burden of data collection Low
Researcher burden of data analysis Moderate
Risk of reactivity bias Depends on blinding
Risk of recall bias No
Risk of social desirability bias No
Risk of observer bias No
Participant literacy required No
Cognitively demanding No

Considerations relating to the use of DLW for assessing physical activity are summarised by population in Table P.3.23.

Table P.3.23 Physical activity assessment by doubly labelled water in different populations.

Population Comment
Pregnancy No special considerations
Infancy and lactation May be difficult to ensure that younger participants have consumed entire dose of enriched water, especially if still being milk fed. Urine can be collected using nappy pads
Toddlers and young children Urine can be collected using nappy pads
Adolescents May require incentives to continue to provide urine samples for entire observation period
Adults No special considerations
Older Adults The method requires urine samples at roughly the same time each day – remembering to do this may be difficult
Ethnic groups No special considerations
Other In obese participants, it may be sensible (and more economical) to base dose weight on estimated total body water
  1. Collaboration with a research group experienced in the DLW technique is strongly recommended.
  2. A ready-made batch dose of DLW i.e. where 2H and 18O have been mixed can be useful when dosing a series of participants.
  3. Batches should be heated in an autoclave for 10 minutes to ensure no bacterial contamination.
  4. A sample of each stock dose should be frozen as the reference for mass spectrometry analysis.
  5. Dosing is usually done on a per kg body weight basis e.g. 18O at 150-174 mg/kg and 2H at 70-80 mg/kg. Alternatively, it can be based on body water.
  1. 2H (deuterium)
  2. 18O
  3. Isotope Ratio Mass Spectrometry facilities.
  4. Sample (urine) collection phials, and if necessary plastic cups and pipettes.
  5. Zipped plastic bag for participants to collect their urine samples in the fridge.
  6. Fridge space at home and freezer space in the laboratory.
  7. Detailed written instructions for participants including a log sheet to record date and time of sample.

A list of specific isotope measurement instruments is being developed for this section. In the meantime, please refer to the overall instrument library page by clicking here to open in a new page.

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