Patients with head and neck cancer (HNC) receiving intensity-modulated radiation therapy (IMRT) have particularly high rates of fatigue, and pre- and post-radiotherapy fatigue are prognostic factors for pathologic tumor responses and poor survival. Although inflammation has been proposed as one of the potential mechanisms of fatigue in cancer patients, findings have not been consistent, and there is a dearth of longitudinal studies. Accordingly, we conducted a prospective study in 46 HNC patients pre- and one-month post-IMRT. Fatigue was measured by the Multidimensional Fatigue Inventory (MFI)-20 at both time points along with the assessment of peripheral blood inflammatory markers including interleukin (IL)-6, soluble tumor necrosis factor receptor 2, and C-reactive protein (CRP) and gene expression. Generalized estimating equations were used to examine the association between inflammatory markers and fatigue. Gene enrichment analysis using MetaCore software was performed using up-regulated genes that were significantly associated with IMRT and fatigue. Significant associations between fatigue and IL-6 as well as CRP, which were independent of time, were observed. In addition the change in fatigue from pre- to post-IMRT was positively associated with the change in IL-6 and CRP. Analysis of up-regulated gene transcripts as a function of IMRT and fatigue revealed overrepresentation of transcripts related to the defense response and nuclear factor kappa B. In conclusion, our findings support the hypotheses that inflammation is associated with fatigue over time in HNC patients. Future studies on how inflammation contributes to fatigue as well as strategies targeting inflammation to reduce fatigue are warranted.