A study was carried out to investigate, whether short-term supplementation of dietary yeast nucleotides affects total and diurnal variation of enzyme activities in the small intestine of weaned piglets. Twelve barrows, weaned at 18 d of age (5 kg initial BW), were fitted with a simple T-cannula at the distal ileum. Twice daily (0730 and 1930 h), 6 piglets each received a cereal-soybean (Glycine max) meal-based diet with or without supplementation of 1 g/kg of a yeast nucleotide product in 2 consecutive periods. In each period, digesta samples were collected 6 times at given intervals during 24 h digesta collection. Dietary supplementation with yeast nucleotides did not affect (P > 0.05) total enzyme activities for α-amylase, leucine aminopeptidase (LAP), maltase, and lactase in ileal digesta. Therefore, data of both treatments were pooled to determine diurnal variations in enzyme activities. For α-amylase, a diurnal variation in enzyme activity could be observed (P < 0.05). Variations in diurnal activities of LAP, maltase, and lactase were not observed (P > 0.05). In conclusion, yeast nucleotides do not affect total small intestinal enzyme activities. Independent of diet composition, α-amylase activities may vary over time, with peak flow at 6 h postprandially.