The demand for healthy products is driving the emerging market of bakery products. The comprehensive evaluation of sourdough fermentation with isolated microbial cultures can provide insights into nutrition and improve technological processes. This study aimed to evaluate the effect of fermentation time on nutritional quality of sourdough breads inoculated with Companilactobacillus paralimentarius 41 (Cp41), Lactiplantibacillus plantarum 47 (Lpb47), Levilactobacillus brevis 83 (Lb83) starter cultures. Three formulations and a non-started dough (control) were developed and fermented for 2, 3, and 4 hours. The pH, total titratable acidity (TTA, mL NaOH 0.1 mol^-1/3g), in vitro protein digestibility (IVPD,%), starch hydrolysis index (HI) and predicted glycemic index (pGI) were determined. At 4th-hour, the pH ranged from 3.85±0.02 to 3.91±0.02, with Lpb47 and Lb83 having the lowest pH. The control had the highest pH (5.56±0.03) and the lowest TTA (1.54±0.05). A significant correlation was observed between pH and the acidifying activity of sourdough LAB during dough fermentation. The IVPD values for sourdough breads were higher at the 4-hour mark, 99.70±0.01 (Lpb47) and 99.30±0.01 (Lb83), compared to the control group (94.40±0.01). This suggests that dough fermentation enhanced the IVPD values, likely due increased proteolytic activity during LAB fermentation. The HI and pGI in sourdough breads increased over time, ranging from 63.48 to 108.77 (Cp41) and 74.56 to 108.77 (Lb83), respectively. The lowest HI values belonged to the Lpb47 and Lb83 bread, similar to the pGI values. The HI increased between 2nd and 4th hours in all sourdough breads. The decrease in resistant starch content could influence in vitro starch digestibility and, consequently, the glycemic response. Fermentation improved nutritional aspects, particularly protein and starch digestibility. Cp41, Lpb47 and Lb83 are promising starter cultures, and further research into the interactions and fermentation conditions of LAB is expected to be investigated.