1. Environmental variability and perturbations can influence population persistence. It is therefore important to understand whether and how animals can compensate for environmental variability, and thereby increase resilience of natural populations. Evolutionary theory predicts that in fluctuating environments selection should favour developmental modifiers that reduce phenotypic expression of genetic variation. The expected result is that phenotypes are buffered from environmental variation across generations. 2. Our aim was to determine whether phenotypes of mosquitofish (Gambusia holbrooki) remain stable across generations in which individuals were born into different thermal environments. We predicted that the spring generation (cool environment) would acclimate by increasing the concentration of regulatory transcription factor mRNA and activities of rate-limiting enzymes (hierarchical regulation) to compensate for the negative thermodynamic effects of lower temperatures on metabolic and locomotor performance. In contrast, the summer-born generation (warm environment) would show less capacity for acclimation and hierarchical regulation. 4. We show that fish from both generations acclimated, but that there were significant differences in the phenotypic consequences of acclimation. The overall result was that sprint performance, metabolic scope, and the activities of cyctochrome c oxidase and lactate dehydrogenase were buffered from environmental change, and did not differ between spring and summer fish at their natural water temperatures of 15oC and 25oC, respectively. However, there were differences between generations in sustained swimming performance and citrate synthase activity. 5. We used metabolic control analysis to show that modes of regulation of metabolic scope and locomotor performance differed between generations. Spring fish showed primarily hierarchical regulation, but regulation in summer fish relied to a lesser extent on rate-limiting enzymes and transcription factors. 6. We suggest that developmental modifiers are favoured in fluctuating environments to maximise phenotypic fitness of each generation. We show that the interaction between developmental and reversible acclimation can render physiological performance of a natural population independent from climate variation.