Particle size alterations during neutral detergent fiber (NDF) determination and in situ rumen incubation were analyzed by dry sieving and image analysis to evaluate the in situ procedure for estimation of NDF degradation parameters and indigestible NDF concentration in terms of particle size. Early-cut and late-cut grass silages, corn silage, alfalfa silage, rapeseed meal, and dried distillers grains were examined. Treatments were (1) drying and grinding of forage samples and grinding of concentrates; (2) neutral detergent-soluble (NDS) extraction; (3) machine washing and NDS extraction; (4) 24-h rumen incubation, machine washing, and NDS extraction; and (5) 288-h rumen incubation, machine washing, and NDS extraction. Degradation profiles for potentially degradable NDF were determined and image analysis was used to estimate particle size profiles and thereby the risk for particle loss. Particle dimensions changed during NDF determination and in situ rumen incubation and variations depended on feedstuff and treatment. Corn silage and late-cut grass silage varied most in particle area among feedstuffs, with an increase of 139% between 0 and 24. h and a decrease of 77% between 24 and 288 h for corn silage and a decrease of 74% for late-cut grass silage between 24- and 288-h in situ rumen incubation. Especially for late-cut grass silage residues after 288 h in situ rumen incubation, a high mass proportion in the critical zone for escape was found. Particle area decreased linearly with increasing incubation time. Particle loss during in situ rumen incubation cannot be excluded and is likely to vary among feedstuffs.