Background There is little consensus on how to measure fatigue. Objectives To standardize the measurement of fatigue across populations, we aimed to assess the psychometric properties of the PROMIS Fatigue item bank in the Dutch general population and obtain reference values. Methods A sample of 1006 people participating in an internet panel completed the full v1.0 PROMIS Fatigue item bank (95 items). Structural validity (item response theory (IRT) assumptions and IRT model fit), measurement invariance/cross-cultural validity (absence of differential items functioning (DIF) for demographic variables and language, compared to data from US participants in PROMIS wave 1), and (internal) reliability (percentage of respondents with reliable estimates) were assessed. Results The IRT model assumptions were considered met (ECV 0.86, Omega-H 0.92), all items fitted the IRT model, no items showed DIF for demographic variables and seven for language, but with negligible impact on T-scores. Reliable fatigue T-scores were found for 98.3%, 69.8–82.6%, and 96.5% of the respondents with the full item bank, the standard short forms, and a simulated computerized adaptive test (CAT), respectively. The CAT administered on average only five items. A T-score of 49.1 represented the average score of the Dutch general population, T-scores <55 are considered within normal limits, T-scores of 55–59 indicate mild fatigue, T-scores of 60–70 indicate moderate fatigue, and T-scores >70 indicate severe fatigue. Conclusions The PROMIS Fatigue item bank showed sufficient structural validity, no measurement invariance for demographic characteristics, sufficient cross-cultural validity, and sufficient (internal) reliability in the Dutch general population.