Comparative physiologists and biochemists working with tissues at varying temperatures and ionic strength are required to adjust apparent equilibrium constants (K′) of biochemical reactions to the experimental conditions prior to calculating cytosolic bioenergetic parameters (transformed Gibbs free energy of formation, _fΔG′_ATP; cytosolic phosphorylation ratio, [ATP]/[ADP][P_i]; [phosphocreatine]:[orthophosphate] ratio [PCr]/[P_i]) and kinetic parameters (free [ADP], [P_i] and [AMP]). The present study shows how to adjust both K′ and the equilibrium constants of reference reactions (K_ref) of creatine kinase (ATP: creatine N-phosphotransferase; EC 2.7.3.2), adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3) and adenosinetriphosphatase (ATP phosphohydrolase; EC 3.6.1.3) to temperature and ionic strength. This information, together with our previous study showing how to adjust equilibria to varying pH and pMg, is vital for the quantification of organ and tissue bioenergetics of ectotherms and endotherms under physiological conditions.