For locally advanced-stage non-small cell lung cancer (NSCLC), inter-fraction target motion variations during the whole time span of a fractionated treatment course are assessed in a large and representative patient cohort. The primary objective is to develop a suitable motion monitoring strategy for pencil beam scanning proton therapy (PBS-PT) treatments of NSCLC patients during free breathing. Methods: Weekly 4D computed tomography (4DCT; 41 patients) and daily 4D cone beam computed tomography (4DCBCT; 10 of 41 patients) scans were analyzed for a fully fractionated treatment course. Gross tumor volumes (GTVs) were contoured and the 3D displacement vectors of the centroid positions were compared for all scans. Furthermore, motion amplitude variations in different lung segments were statistically analyzed. The dosimetric impact of target motion variations and target motion assessment was investigated in exemplary patient cases. Results: The median observed centroid motion was 3.4 mm (range: 0.2-12.4 mm) with an average variation of 2.2 mm (range: 0.1-8.8 mm). Ten of 32 patients (31.3%) with an initial motion <5 mm increased beyond a 5-mm motion amplitude during the treatment course. Motion observed in the 4DCBCT scans deviated on average 1.5 mm (range: 0.0-6.0 mm) from the motion observed in the 4DCTs. Larger motion variations for one example patient compromised treatment plan robustness while no dosimetric influence was seen due to motion assessment biases in another example case. Conclusions: Target motion variations were investigated during the course of radiotherapy for NSCLC patients. Patients with initial GTV motion amplitudes of < 2 mm can be assumed to be stable in motion during the treatment course. For treatments of NSCLC patients who exhibit motion amplitudes of > 2 mm, 4DCBCT should be considered for motion monitoring due to substantial motion variations observed.