Sugars are known to improve neutral detergent fiber (NDF) digestibility in lactating dairy cows. However, not much information is known about specific monosaccharides and inclusion levels. A study was conducted to investigate the effects of glucose, fructose, xylose and rhamnose on in vitro NDF digestibility (NDFd), pH and VFA concentration. The sugars were selected from a preliminary trial. Either oat hay (OH), alfalfa hay (AH) or a alfalfa hay-based TMR were separately fermented in vitro with each sugar for 12 and 24 h using different inclusion levels: 0% (control), 10%, 15% or 20% of the NDF content of the feed samples used. Combinations of the samples and the sugars were run in triplicates, across 3 runs. Data were analyzed according to a randomized complete block design with a factorial arrangement of treatments. Each sugar was analyzed separately, with level, feed sample and their interaction as fixed effects and run as random factor. The addition of fructose and rhamnose increased NDFd at the 15% and 20% inclusion levels (P < 0.05), respectively, while glucose and xylose were significant only at 10% of the NDF (P = 0.035 and P = 0.022, respectively). Overall, the sugars were more effective in increasing NDFd when added to the TMR (P < 0.05), followed by LH and OH. Independently of the sugar and sample used, the in vitro system was able to maintain the pH above 6.8 (P > 0.05) across sugars and inclusion levels. The main VFA quantified increased when sugars were added (P < 0.05), especially with the highest inclusion level (P < 0.05). Interestingly, while acetic acid was variable, both propionate and butyrate were highest with 20% inclusion level for all sugars (P < 0.05). This preliminary work demonstrates the need to explore the effects of specific monosaccharides addition on diets of dairy cows as a way of improving the rumen microenvironment.