Undigested forage NDF residues (uNDF) from long-term ruminal in situ incubations are used to estimate indigestible fiber (iNDF). Estimation of iNDF is important in forage evaluation because it defines the potentially digestible pool of NDF. Near-infrared reflectance spectroscopy (NIRS) can be calibrated to in situ reference sets to rapidly predict uNDF. Our objective was to compare uNDF estimates after 240 h of incubation when 2 types of bags were used in the in situ reference method. The bags compared were 4 cm × 5 cm Ankom F57 bags (25-μm pore size), and 5 cm × 10 cm Ankom in situ bags (50-μm pore size). Alfalfa samples taken from Pennsylvania and Wisconsin (n = 144) of different varieties and harvest intervals were used. Forages were dried 48h at 60°C and ground through a 2mm screen in Wiley mill. One-half or 2 g samples, respectively, were weighed into the small and large bags in triplicate. Mass to surface area was 0.05 and 0.02 g/cm² for the small and large bags, respectively. The uNDF content after 240 h incubation were evaluated by 2 bag types in 3 rumen-cannulated Holstein cows. Each dried and ground forage was also scanned to determine the visible–near-infrared-reflectance spectra using with a FOSS NIRSystems 6500 spectrophotometer. Prediction equations were developed for each bag type using partial least square regressions. The estimated uNDF fraction from small and large bags were 13.75% and 9.97%, respectively (SED = 0.39, P < 0.001). The coefficient of determination for calibration (RSQ), cross-validation (1 − VR), calibration standard deviation (SEC), and interactive authentication standard deviation (SECV) was 0.94, 0.92, 0.85 and 0.98 for uNDF values determined with the small bag and 0.88, 0.85, 1.12 and 1.27 for uNDF values determined with the large bag calibration sets, respectively. Results indicate that uNDF vary and NIRS can be used to quickly and quantitatively estimate iNDF content in alfalfa. Bag type influences 240-h NDF residues. NIRS predictions of uNDF from the small bag calibration had higher RSQ and lower SEC and SECV than the large bag calibrations.