Background: Flexion-rotation test predominantly measures rotation in C1-2 segment. Restriction in flexion-rotation may be due to direct limitation in C1-2, but also to a premature tightening of the alar ligament as a result of lack of movement in C0-1 or C2-3. The aim of this study was to compare the effect of a 20-min single cervical exercise session, with or without manual therapy of C0-1 and C2-3 segment in flexion-rotation test, in patients with chronic neck pain and positive flexion-rotation test. Methods: Randomized controlled clinical trial in 48 subjects (24 manual therapy+exercise/24 exercise). Range of motion and pain during flexion-rotation test, neck pain intensity and active cervical range of motion were measured before and after the intervention. Results: Significant differences were found in favour of the manual therapy group in the flexion-rotation test: right (p < 0.001) and left rotation (p < 0.001); pain during the flexion-rotation test: right (p < 0.001) and left rotation (p < 0.001); neck pain intensity: (p < 0.001); cervical flexion (p < 0.038), extension (p < 0.010), right side-bending (p < 0.035), left side-bending (p < 0.002), right rotation (p < 0.001), and left rotation (p < 0.006). Conclusions: Addition of one C0-C1 and C2-C3 manual therapy session to cervical exercise can immediately improve flexion-rotation test and cervical range of motion and reduce pain intensity.