Temperature dependent adjustments of intracellular pH are thought to play a major role in the maintenance of protein function. Comparative studies were carried out in two species from the same fish family (Zoarcidae), the stenothermal Antarctic eelpout (Pachycara brachycephalum) and the eurythermal eelpout (Zoarces viviparus) to find out whether pH regulation is modified by temperature in both closely related species and to what extent the respective pattern differs between eurytherms and stenotherms. Previous invasive studies had compared individual animals sampled at various temperatures thereby suggesting that a decrease in intracellular pH (pHi) values occurs at rising temperatures as predicted by the alpha-stat hypothesis of acid-base regulation. The present study used non-invasive in vivo 31P-NMR spectroscopy in non-anaesthetized, unrestrained fish for long term online recordings in individual specimens. Control spectra obtained at T= 0 °C for P. brachycephalum and at 12 °C for Z. viviparus indicated low stress conditions as well as a high stability of energy and acid-base status over time periods longer than 1 week. Temperature changes had no influence on the concentration of high-energy phosphates like phosphocreatine or ATP. Temperature induced pH changes were monitored continuously in a range between 0 and 6 °C for polar and 12 to 18 °C for temperate eelpout. A pHi change of around -0.015 pH units/°C was observed within both species in accordance with the alpha-stat hypothesis, however, extrapolation to the same temperature revealed different set points of pH regulation in the two species. These findings confirm that an alpha-stat pattern of pH regulation can be found in stenothermal Antarctic animals, however, at setpoints deviating from an alphastat pattern in a between species comparison.