A central issue in the use of probiotics in food and food supplements is their sensitivity to many environmental stress factors. The resistance of probiotic cells to lethal stress can be improved by application of homologous or heterologous sub-lethal stress during culture. This screening procedure is generally performed using batch cultures. Continuous cultures could be a suitable and more efficient method to test different stress factors on one culture instead of repeating several batch cultures. However, before testing stresses using continuous cultures, the physiological stability of continuously produced cells over a considered time period must be first evaluated. A continuous culture of Bifidobacterium longum NCC2705 was maintained for 211 h at a dilution rate of 0.1 per h, mimicking a deceleration growth phase culture. Stable viable cell counts were measured over the culture period, decreasing only moderately from 8.8 to 8.6 log10 cfu/ml. A slight shift in metabolite production, characterized by increased lactate and decreased acetate, formate and ethanol concentrations was observed. Susceptibilities to antibiotics and stress conditions were stable (cefotaxim, ampicillin, ceftazidime) or moderately affected (simulated gastric juices, heat, bile salts, tetracycline, chloramphenicol, penicillin, vancomycin and neomycin) over culturing time. Comparison of gene transcription profiles between samples collected after 31 h of continuous culture and samples collected after 134 and 211 h revealed only limited changes in expression of 1.0 and 3.8% of total genes, respectively. Based on these results, we propose that continuous culture can be used to produce bacterial cells with stable physiological properties suitable for fast and efficient screening of sub-lethal stress conditions.