Experimental unit

The experimental unit is the entity subjected to an intervention independently of all other units. It must be possible to assign any two experimental units to different treatment groups.


The experimental unit is the entity a researcher wants to make inferences about (in the population) based on the sample (in the experiment). Thus this is the entity for which adequate replication is needed. The sample size is the number of experimental units per group. If the experimental unit is not identified correctly, there is a risk that the n number is over-estimated which might invalidate the result of the statistical analysis and the conclusions.

Commonly the experimental unit is the individual animal and each animal is allocated to a particular treatment group independently of other animals. Occasionally, depending on the treatment administered, the experimental unit may be bigger than the animal (e.g. a litter or a cage) or smaller than the animal (e.g. part of the animal, an animal for a period of time). Below are various examples of experimental units.

Note that taking multiple measures from the same animal does not mean that each animal provides multiple experimental units. The experimental unit is defined as the entity which receives an intervention or treatment, regardless of how many measurements it is subjected to.

On the EDA diagram, the experimental unit node is connected from one of the group nodes as shown in the image below.

Only one experimental unit node per diagram is necessary if the experimental unit is the same throughout the experiment, if there are multiple experimental units, multiple nodes may be necessary to clarify what unit different interventions are applied to.

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Examples of experimental units

The individual animal

This is the most common situation and individual animals are independently assigned to distinct categories of the variable(s) of interest. It must be possible for any two individuals to receive different treatments. For example in Example 4, the two variables of interest which define the four groups are sex and exercise. Animals are either male or female independently of other animals, and they are also allocated to different activity levels independently of other animals. Thus the experimental unit is the individual mouse. 

A breeding female and litter 

Consider a teratogenesis experiment where the pregnant female receives a treatment and measurements are made on the individual pup after birth. Animals within a litter are all exposed to the same treatment thus the experimental unit is the whole litter. In this case, the variable ‘individual pups’ is nested into the experimental unit ‘litter’.

The cage of animals 

If animals are group housed in a cage and all animals within that cage receive the same treatment, for example in the drinking water or diet, then the experimental unit is the cage of animals. If animals are group housed but can each receive a different treatment, for example by injection, then the experimental unit would be the individual animal.

A part of an animal

If animals are exposed to a treatment via topical application, it may be possible to divide an area of skin into a number of different patches which can each receive distinct treatments. In this situation, the patch of skin on the animal is the experimental unit.

If individual cells can be stimulated independently and recording of the responses is made at the individual cell level, the experimental unit for the stimulation is the individual cell. Provided the experiment does not include another treatment which the whole animal is exposed to (e.g. drug injection or genotype), the individual cell can be the experimental unit for the whole experiment and a single animal provides many experimental units. If just a single animal is used, then the results hold true for that animal alone and cannot be generalised to the population.

When a single animal provides multiple experimental units, to avoid the confounding effect of between-animal variability, the individual animal should be used as a blocking factor.

An animal for a period of time

In a crossover experiment where each animal is used as its own control and receives distinct treatments, separated by wash out periods, each animal provides several experimental units as animals can be exposed to different treatments in different test periods. The experimental unit is thus the animal for period of time. This is exemplified in Example 2.

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More than one type of experimental unit in an experiment 

Occasionally, there may be multiple experimental units in a single experiment, for example in a so called split plot or a nested experiment.

Consider a situation where the effects of two different treatments (diet and vitamin supplements) on growth rate are investigated in mice. Diet is administered at the cage level and all mice housed in the same cage receive the same diet; thus the experimental unit for the diet treatment is the cage. However, the vitamin supplement is administered by gavage, thus animals within the same cage can receive different supplements and the experimental unit for the effect of vitamin supplement is the individual mouse.

This type of design is powerful as it enables researchers to investigate whether the effect of the vitamin is related to the diet administered; however the statistical analysis of such designs can be complicated and expert statistical advice should be sought before conducting such an experiment.

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References and further reading

FESTING, M. F. W., OVEREND, P., GAINES DAS, R., CORTINA BORJA, M. & BERDOY, M. 2002. The design of animal experiments: reducing the use of animals in research through better experimental design, London UK, Royal Society of Medicine.

FESTING, M. F., http://www.3rs-reduction.co.uk/html/3__the_experimental_unit.html. [Accessed 15-01-2015]