The purpose of this study was to investigate the effects of smartphone use on muscle fatigue and tenderness in the cervical erector spinae (CES) and the upper trapezius (UT) and on the cervical range of motion among subjects with and without neck muscle pain. The subjects were 30 smartphone users in their 20 s who -were assigned to either an experimental group with neck muscle pain or a control group without neck muscle pain. Muscle fatigue and tenderness in the CES and the UT as well as the subjects’ cervical range of motion were measured before and after 20-min smartphone sessions in a sitting position. In a between-group comparison of muscle fatigue, the experimental group showed a significantly greater decrease in median frequency in the CES and the right UT after smartphone use (p<.05). Regarding the assessment of muscle tenderness after smartphone use, the experimental group showed a statistically significant decrease in the pressure-pain threshold (PPT) in all muscles (p<.05), whereas the control group showed a significantly decreased PPT in the right CES and the UT (p<.05). The assessment of the cervical range of motion revealed a statistically significant reduction in the cervical flexion-extension and left lateral flexion in the experimental group (p<.05) after smartphone use. However, there was no significant change in the cervical range of motion in the control group (p>.05) after smartphone use. When compared with the control group, the experimental group demonstrated greater changes in cervical extension, lateral flexion, and rotation, except for cervical flexion (p<.05). In conclusion, when smartphone users have pre-existing neck muscle pain, the use of a smartphone further increased muscle fatigue and tenderness in the neck and reduced PPT and the cervical range of motion.