This study investigated the between trial variation of thermoregulatory measures during a heat acclimation protocol. Eight 14-16 y old boys completed three bouts of 20-min cycling at 45 % peak VO₂ in a hot environment (35.1 ± 1.2 °C and 46. 4 ± 1.0 % relative humidity) on two occasions separated by a minimum of 24 h. Reliability was assessed through analysis of within-subject variation, the change in the mean, and retest correlation for measurements of aural temperature (T_au), mean skin temperature (T_sk), heart rate (HR) and oxygen uptake (VO₂). Between trial differences were low for T_au, T_skbout1, T_skbout2and3 and HR with coefficients of variation 0.6 %, 1.5 %, 0.5 % and 4.0 %, respectively. The results demonstrate good reliability that will allow future investigators to precisely determine the extent of heat acclimation protocols in relation to the measurement error.

• To allow paediatric heat acclimation guidelines to be produced, the extent of heat acclimation needs to be established once biological and mechanical variation has been accounted for.

• The results of the present study indicate that between trial differences were low for aural temperature, mean skin temperature and heart rate with coefficient of variation values ranging from 0.6 - 4.0 %.

• Future investigators will be able to utilise the coefficient of variation values to establish the effectiveness of heat acclimation with precision alongside the selection of appropriate sample size.