Physical exertion leads to the rise in tear osmolarity. However, previous studies have been conducted mostly on males and did not consider sex differences and the possible alteration in blinking during physical exercise. Sixteen women and 18 men aged 25.09 ± 1.70 were divided into equal groups with eyes open and shut. Participants performed 8-min medium-intensity exercise and 5-min intense exercise on a cycloergometer. Tear osmolarity (in mOsm/L) was evaluated before ( T0), after medium-intensity (T1) and intense exercise (T2). The blinking rate was assessed in a group with eyes open. Tear brake up time was measured in T0 and T1. With tear osmolarity measuring 305.72 ± 1.22 and 313.56 ± 1.90 for men and women, respectively, we observed significant differences in T1. In T2, tear osmolarity in men was 303.3 ± 1.28 vs. 310.87 ± 1.36 in women. The blinking rate decreased from 14.24 ± 2.54/min in T0 to 9.41 ± 2.83/min in T1. There was a statistically significant change in tear osmolarity in both groups, that is, in the group with eyes shut from 300.53 ± 1.37 in T0 to 308.06 ± 1.55 in T1 to 304.88 ± 1.54 in T2. In the group with eyes open, tear osmolarity increased from 300.29 ± 1.37 in T0 to 310.76 ± 1.55 in T1 and then dropped to 308.88 ± 1.54 in T2. Tear brake up time measured in T0 was 14.7 ± 1.43 vs. 13.53 ±1.48 in the open eyes condition. Due to physical exercise, short-term changes in tear osmolarity are partially caused by altered blinking. Sex differences in tear osmolarity in response to exertion may confirm the relationship between total body water and tear osmolarity.