Many invasive species have evolved behavioural and morphological characteristics that facilitate their dispersal into new areas, but it is unclear how selection on this level of the phenotype filters through to the underlying physiology. Cane toads have been dispersing westward across northern tropical Australia for more than 70 years. Previous studies of cane toads at the invasive front have identified several behavioural, morphological and locomotory characteristics that have evolved to facilitate dispersal of toads. We assessed a range of physiological characteristics associated with locomotory abilities in toads from the long-established, east coast of Australia, from the invasive front, and from a site in between these locations. We measured time to exhaustion and respiratory gases of toads exercising on a treadmill, time to recovery from exhaustion, blood properties (lactate, haematocrit, haemoglobin, red blood cell count, blood cell volume), and muscle properties associated with locomotion (activities of the enzymes citrate synthase and lactate dehydrogenase, and pH buffering capacity). None of the measured physiological parameters supported the hypothesis that toads from the invasive front possess physiological adaptations that facilitate dispersal compared to toads from areas colonised in the past. The strongest difference among the three groups of toads, time to exhaustion, showed exactly the opposite trend; toads from the long-established populations in the east coast had the longest time to exhaustion. Successful colonisers can employ many characteristics to facilitate their dispersal, so the extent to which behaviour, morphology and physiology co-evolve remains an interesting question. However, in the present case at least, behavioural adaptations do not appear to have altered the organism's underlying physiology.