The improvement of in vitro digestion models is essential and depends, among others, on a better knowledge of the enzymatic activities in the digestive tract of humans. Within the framework of a collaboration between INFOGEST and UNGAP, a cost action on oral drug absorption, access to intestinal fluids was possible. Human intestinal fluids were collected under fasting and fed state conditions using a catheter placed in the duodenum. Intestinal fluids from 13 healthy adult volunteers were used for the determination of digestive enzyme activities. Intestinal fluids were collected kinetically over 110 min after ingestion of 240 ml of water (fasted) or 400 ml of Ensure Plus vanilla followed 20 min after by 240 mL of water (fed), leading to a total of 88 samples. Immediately after collection, intestinal fluids were mixed with glycerol (1:1) and inhibitors to protect digestive enzymes from enzymatic breakdown. Then, trypsin, chymotrypsin, and lipase activities were determined using the enzymatic assays protocols described in Brodkorb et al. (2019), except that the temperature for the trypsin assay was 37°C. The pH of each sample was also measured immediately after collection. Mean values of 64.9 ± 28.5, 24.2 ± 10.2 and 389 ± 253 Units /ml of duodenal content (fasted) and 62.8 ± 28.9, 22.9 ± 9.6 and 1071 ± 60.7 Units /ml of duodenal content (fed) were obtained for trypsin, chymotrypsin and lipase, respectively. Mean pH was 6.3 ± 1.3 and 6.1 ± 0.8 under fasted and fed state conditions, respectively. No effect of nutritional state (fasted vs fed) on the enzymatic nor on the pH was observed. Due to a high inter-individual variability between volunteers, no effect of the time of sampling was observed on the activity level of trypsin, chymotrypsin and lipase in the duodenum. Few data are currently available in the literature and this new set of data is of major importance. More in vivo data on enzymes activities in the upper part of the gastro-intestinal tract in different prandial states would be needed to help the scientific community improving in vitro digestion models.