The calculations for the net gain of ATP is usually based on the following assumptions :

i.  All the reactions are sequential and follow a orderly pathway , with one substrate forming the next and with glycolysis, TCA cycle and ETS
pathway following one after another.

ii. The NADH synthesised in glycolysis is transferred into the mitochondria where it is subject to oxidative phosphorylation.

iii. The intermediates of the pathway are not utilised to synthesise any other compound.

iv. Only glucose is used as the respiratory substrate and no alternative r alternative substrates are used or enter the pathway at any of the intermediary stages.