What are the assumptions made during the calculation of net gain of ATP?

It is assumed that glycolysis, the TCA cycle, and the electron transport system occur in a single, sequential, orderly pathway without overlap

NADH produced during glycolysis is assumed to be fully transported into mitochondria (via shuttles) and entirely available for oxidative phosphorylation

Glucose is considered the sole respiratory substrate, with no other molecules entering the pathway at intermediate stages

No intermediates are withdrawn for biosynthetic or other processes; all stay within the respiratory pathway

A fixed P/O ratio is assumed, typically 3 ATP per NADH oxidized and 2 ATP per FADH₂ oxidized

Proton translocation and ATP synthesis are assumed to be completely coupled, with no proton leak or uncoupling reducing efficiency

No ATP is consumed for transport of ADP, Pi, or ATP across the mitochondrial inner membrane (e.g., by the adenine nucleotide translocase)