Abstract: Accurate estimates of bacterial carbon metabolic rates are indispensable for understanding the regulation of carbon fluxes in aquatic environments. Here, changes in bacterial growth, production, and cell volume in both pre-filtered and unfiltered seawater during 24 h incubation were monitored. The methodological artifacts during Winkler bacterial respiration (BR) measurements in subtropical Hong Kong coastal waters were assessed. Bacterial abundance increased by 3- and 1.8-fold in the pre-filtered and unfiltered seawater after incubation, respectively. Bacterial production (BP) and cell volume also showed significant enhancement. Compared with the BR measurements obtained by the Winkler method, the instantaneous free-living BR measurements, after correction, decreased by ~ 70%. The time-integrated free-living BR and BP during 24 h incubation in the pre-filtered sample provided an improved estimate of bacterial growth efficiency, which increased by ~ 52% compared to the common estimations using the noncomparable measurements of integrated free-living BR and instantaneous total BP. The overestimation of BR also exaggerated the contribution of bacteria to community respiration, affecting the understanding on the metabolic state of the marine ecosystems. Furthermore, the BR estimates by the Winkler method may be more biased in environments with a higher bacterial growth rate and tightly coupled grazing mortality, as well as in those with higher nutrient concentrations. These results reveal obvious problems associated with the BR methodology and raise a warning for caution when comparing BP and BR, as well as when making estimations of carbon flow through the complex microbial networks in aquatic ecosystems.