Fish were sampled throughout the diel temperature cycle according to the following schedule: (i) Tmin following the ‘intermediate’ temperature increase (t = 48 h); (ii) Tmax, the first day of heat cycling (t = 60 h); (iii) the first Tmin throughout heat cycle (16°C, 18°C, 20°C or 22°C, depending on treatment, t = 72 h); (iv) Tmax, the third day of heat cycling (t = 108 h); and (v) Tmin, the third day of heat cycling (t = 120 h, Fig. 1B). These sampling time points were chosen to establish the physiological condition prior to heat exposure, the implications of 1 day of heat cycling, and the potential cumulative effect of 3 days of cycling. This 5-timepoint sampling regime was performed on all treatment groups. However, to address the potential effect of ‘trial’ on our results, two additional sampling time points (t = 12 h and t = 24 h) were established in one treatment group in each of the two trials (16–21°C for Trial 1 and 18–27°C for Trial 2), prior to the heat cycle. This additional sampling allowed us to further address the incipient, ‘pre-thermal stress’ condition.