The assumptions made during the calculation of ATP are :

(a) The reaction like glycolysis, TCA cycle, and ETS, are assumed to occur in a sequential and orderly pathway.

(b) NADH produced during the process of glycolysis enters into mitochondria to undergo oxidative phosphorylation.

(c) Glucose is assumed to be the only substrate

(d) It is assumed that no other molecule enters the pathway at the intermediate stages.

(e) The intermediates produced during respiration are not utilized in any other process.