Oxygen-balanced mixotrophy (OBM) is a novel type of microalgal cultivation that improves autotrophic productivity while reducing aeration costs and achieving high biomass yields on substrate. The scale-up of this process is not straightforward, as nonideal mixing in large photobioreactors might have unwanted effects in cell physiology. We simulated at lab scale dissolved oxygen and glucose fluctuations in a tubular photobioreactor operated under OBM where glucose is injected at the beginning of the tubular section. We ran repeated batch experiments with the strain Galdieria sulphuraria ACUF 064 under glucose pulse feeding of different lengths, representing different retention times: 112, 71, and 21 min. During the long and medium tube retention time simulations, dissolved oxygen was depleted 15-25 min after every glucose pulse. These periods of oxygen limitation resulted in the accumulation of coproporphyrin III in the supernatant, an indication of disruption in the chlorophyll synthesis pathway. Accordingly, the absorption cross-section of the cultures decreased steeply, going from values of 150-180 m 2 kg −1 at the end of the first batch down to 50-70 m 2 kg −1 in the last batches of both conditions. In the short tube retention time simulation, dissolved oxygen always stayed above 10% air saturation and no pigment reduction nor coproporphyrin III accumulation were observed. Concerning glucose utilization efficiency, glucose pulse feeding caused a reduction of biomass yield on substrate in the range of 4%-22% compared to the maximum levels previously obtained with continuous glucose feeding (0.9 C-g C-g −1 ).The missing carbon was excreted to the supernatant as extracellular polymeric substances constituted by carbohydrates and proteins. Overall, the results point out the importance of studying large-scale conditions in a controlled environment and the need for a highly controlled glucose feeding strategy in the scale-up of mixotrophic cultivation.