Abstract: In order to investigate the puff-by-puff release of main components in aerosols of heat-not-burn (HnB) tobacco products, samples of two commercial HnB tobacco products (IQOS and GLO) with different heating methods were studied with puff-by-puff puffing and aerosol trapping via a modified rotary smoking machine. A method for detecting the glycerol, nicotine, water content and main aroma substances in the HnB aerosols was established, and the puff-by-puff variations of main aerosol components were measured under two puffing regimes (ISO and HCI). The results showed that:1) The average recovery of nicotine, glycerol and water contents was higher than or equal to 99.7%, and the relative standard deviation was less than or equal to 5.32%. 2) Under the two puffing regimes, the releases of nicotine and glycerol in IQOS aerosols increased first, then stabilized and finally decreased with the proceeding puffs. The release of nicotine in GLO aerosols was more variable, it was increased and then decreased in the first few puffs, then increased and finally decreased again with the remaining puffs, while the glycerol level in GLO increased first and then decreased. 3) With the proceeding puffs, the water content in IQOS aerosols increased first and then decreased under ISO puffing regime, while gradually decreased under HCI puffing regime. The water content in GLO aerosols gradually decreased under the two smoking regimes. 4) With the proceeding puffs, the neophytadiene in IQOS aerosols increased gradually under ISO smoking regime, while increased first and then decreased under HCI smoking regime. The levels of furfural, hydroxy acetone, acetic acid and solanone increased first and then decreased, while 5-methylfurfuraldehyde and furfuryl alcohol levels increased gradually under the two puffing regimes. The main aroma substances in GLO aerosols presented a double increasing first and then decreasing trend under the two puffing regimes.